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WO2017177137A1 - Compositions de lymphocytes t récepteurs d'antigènes chimériques - Google Patents

Compositions de lymphocytes t récepteurs d'antigènes chimériques Download PDF

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Publication number
WO2017177137A1
WO2017177137A1 PCT/US2017/026602 US2017026602W WO2017177137A1 WO 2017177137 A1 WO2017177137 A1 WO 2017177137A1 US 2017026602 W US2017026602 W US 2017026602W WO 2017177137 A1 WO2017177137 A1 WO 2017177137A1
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Prior art keywords
cell
car
cells
domain
tcra
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PCT/US2017/026602
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English (en)
Inventor
Michael T. Certo
Wai-Hang LEUNG
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Bluebird Bio, Inc.
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Application filed by Bluebird Bio, Inc. filed Critical Bluebird Bio, Inc.
Priority to CA3020330A priority Critical patent/CA3020330A1/fr
Priority to AU2017248259A priority patent/AU2017248259A1/en
Priority to US16/092,146 priority patent/US20190161530A1/en
Priority to CN201780031902.3A priority patent/CN109415687A/zh
Priority to JP2018552793A priority patent/JP2019510503A/ja
Priority to EP17779910.3A priority patent/EP3440191A4/fr
Publication of WO2017177137A1 publication Critical patent/WO2017177137A1/fr

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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C—CHEMISTRY; METALLURGY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
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    • A—HUMAN NECESSITIES
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to improved immune effector cell compositions for adoptive cell therapy. More particularly, the invention relates to improved chimeric antigen receptor (CAR) T cell compositions, and method of making and using the same.
  • CAR chimeric antigen receptor
  • Cancer The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer related deaths in 2012.
  • the most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer.
  • the number of new cancer cases is projected to rise to 22 million within the next two decades.
  • the invention generally relates, in part, to improved immune effector cell compositions and methods of manufacturing the same.
  • the immune effector cells contemplated in particular embodiments comprise a chimeric antigen receptor (CAR) and a precise disruption or modification in one or more T cell receptor loci, which leads to disruption of TCR expression and signaling.
  • CAR T cells comprising one or more modified T cell receptor alpha (TCRa) alleles secrete increased amounts of cytolytic cytokines and are more therapeutically efficacious than CAR T cells that lack a modified TCRa allele.
  • TCRa modified T cell receptor alpha
  • a chimeric antigen receptor (CAR) T cell comprising, one or more modified T cell receptor alpha (TCRa) alleles, wherein the CAR T cell secretes an increased amount of proinflammatory cytokines when bound to a target antigen compared to a CAR T cell bound to the target antigen that lacks one or more modified TCRa alleles.
  • CAR modified T cell receptor alpha
  • the CAR comprises an extracellular domain that binds a target antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72.
  • the CAR comprises a transmembrane domain isolated from a polypeptide selected from the group consisting of: alpha or beta chain of the T-cell receptor,
  • the CAR comprises one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-lBB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70.
  • a polypeptide selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-lBB), CD278 (ICOS), DAP10, LAT, KD2C, SLP
  • the CAR comprises a signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRP, CD3y, CD35, CD3s, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • the CAR comprises a hinge region polypeptide selected from the group consisting of: a hinge region of CD8a, a hinge region of PDl, and a hinge region of CD152.
  • the CAR comprises one or more linker polypeptides.
  • the CAR comprises a spacer region polypeptide that comprises CH2 and CH3 regions of IgGl, IgG4, or IgD.
  • the CAR comprises a signal peptide selected from the group consisting of: an IgGl heavy chain signal polypeptide, a CD8a signal polypeptide, and a human GM-CSF receptor alpha signal polypeptide.
  • the one or more modified TCRa alleles are non-functional or have substantially reduced function.
  • the CAR T cell comprises one or more proviral integrants comprising a nucleic acid encoding the CAR.
  • the CAR T cell comprises a polynucleotide comprising a promoter operably linked to a nucleic acid encoding the CAR, wherein the polynucleotide has been inserted into the one or more TCRa alleles by homology directed repair.
  • the one or more proinflammatory cytokines are selected from the group consisting of: ⁇ , TLA, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and ⁇ >- la.
  • a CAR T cell comprising: one or more modified TCRa alleles; and a nucleic acid encoding a CAR that binds a target antigen, wherein the CAR T cell secretes an increased amount of one or more proinflammatory cytokines when contacted with a target cell expressing the target antigen compared to a CAR T cell bound to the target antigen that lacks one or more modified TCRa alleles.
  • a CAR T cell comprising: one or more modified TCRa alleles; and one or more proviral integrants comprising a nucleic acid encoding a CAR that binds a target antigen, wherein the CAR T cell secretes an increased amount of one or more proinflammatory cytokines when bound to a target antigen compared to a CAR T cell bound to the target antigen that lacks one or more modified TCRa alleles.
  • a CAR T cell comprising: one or more modified TCRa alleles, wherein a donor repair template comprising a nucleic acid encoding a CAR that binds a target antigen is inserted into the one or more TCRa alleles by homology directed repair, and wherein the CAR T cell secretes an increased amount of one or more
  • proinflammatory cytokines when bound to a target antigen compared to a CAR T cell bound to the target antigen that lacks one or more modified TCRa alleles.
  • the nucleic acid further comprises an RNA polymerase ⁇ promoter operably linked to the polynucleotide encoding the CAR.
  • the RNA polymerase ⁇ promoter is selected from the group consisting of: a short EFla promoter, a long EFla promoter, a human ROSA 26 locus, a Ubiquitin C (UBC) promoter, a phosphogly cerate kinase- 1 (PGK) promoter, a
  • CAG cytomegalovirus enhancer/chicken ⁇ -actin
  • MND myeloproliferative sarcoma virus enhancer
  • the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72.
  • the CAR further comprises a transmembrane domain and one or more intracellular signaling domains. In further embodiments, the CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain.
  • the CAR further comprises a signal peptide, one or more linker peptides, and one or more hinge peptides, and optionally one or more spacer peptides.
  • the CAR comprises an anti-BCMA scFv, a CD8a transmembrane domain, a CD137 costimulatory domain, and a 0)3 ⁇ primary signaling domain.
  • the CAR comprises an anti-CD 19 scFv, a CD8a
  • transmembrane domain a CD137 costimulatory domain
  • a 0)3 ⁇ primary signaling domain a CD137 costimulatory domain
  • the CAR comprises an anti-CSPG4 scFv, a CD8a
  • transmembrane domain a CD137 costimulatory domain
  • a 0)3 ⁇ primary signaling domain a CD137 costimulatory domain
  • the CAR comprises an anti-RORl scFv, a CD8a transmembrane domain, a CD137 costimulatory domain, and a 0)3 ⁇ primary signaling domain.
  • the CAR comprises an anti-PSCA scFv, a CD8a
  • transmembrane domain a CD137 costimulatory domain
  • a 0)3 ⁇ primary signaling domain a CD137 costimulatory domain
  • the CAR comprises an anti-TAG72 scFv, a CD8a transmembrane domain, a CD137 costimulatory domain, and a 0)3 ⁇ primary signaling domain.
  • the one or more proinflammatory cytokines are selected from the group consisting of: ⁇ , IL-4, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and ⁇ >- la.
  • composition comprising a CAR T cell contemplated herein.
  • a composition comprising a CAR T cell contemplated herein and a physiologically acceptable excipient.
  • a method of making a CAR T cell comprising: engineering a double-strand break at a target site in a TCRa allele, wherein the break is repaired by non-homologous end joining (HEJ), thereby generating a modified TCRa allele; and transducing the T cell with a viral vector encoding a CAR; wherein the CAR T cell secretes an increased amount of one or more proinflammatory cytokines when bound to a target antigen compared to a CAR T cell bound to the target antigen that lacks a modified TCRa allele.
  • HEJ non-homologous end joining
  • the modified TCRa allele is non-functional or has substantially reduced function.
  • the viral vector is a retroviral vector.
  • the retroviral vector is a lentiviral vector.
  • a method of making a CAR T cell comprising: engineering a double-strand break at a target site in a TCRa allele; and transducing the T cell with a viral vector comprising a donor repair template that comprises an RNA polymerase II promoter operably linked to a nucleic acid encoding a CAR; wherein the donor repair template is incorporated into the TCRa allele by homology directed repair at the site of the double- strand break (DSB); and wherein the CAR T cell secretes an increased amount of one or more proinflammatory cytokines when bound to a target antigen compared to a CAR T cell bound to the target antigen that lacks a modified TCRa allele.
  • a viral vector comprising a donor repair template that comprises an RNA polymerase II promoter operably linked to a nucleic acid encoding a CAR
  • the donor repair template is incorporated into the TCRa allele by homology directed repair at the site of the double- strand break (DSB)
  • the donor repair template comprises a 5 ' homology arm homologous to the TCRa sequence 5 ' of the DSB; and a 3 ' homology arm homologous to the TCRa sequence 3 ' of the DSB.
  • the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.
  • the rAAV has one or more ITRs from AAV2.
  • the rAAV has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.
  • the retrovirus is a lentivirus.
  • the lentivirus is an integrase deficient lentivirus.
  • the double-strand break is generated with an engineered nuclease.
  • the engineered nuclease is selected from the group consisting of: a meganuclease, a megaTAL, a TALEN, a ZFN, or a CRISPR/Cas nuclease.
  • the meganuclease is engineered from an I-Onul LHE.
  • the engineered nuclease is a megaTAL comprising a TALE DNA binding domain and an engineered meganuclease.
  • the double-strand break is generated by an engineered endonuclease and an end-processing enzyme.
  • an mRNA encoding the engineered endonuclease and an mRNA encoding the end-processing enzyme are introduced into the T cell to generate the double-strand break.
  • an mRNA encoding the engineered endonuclease, a viral- self cleaving peptide, and an end-processing enzyme are introduced into the T cell to generate the double-strand break.
  • an mRNA encoding the engineered endonuclease, an IRES element, and an end-processing enzyme are introduced into the T cell to generate the double- strand break.
  • an mRNA encoding the engineered endonuclease fused to an end-processing enzyme or biologically active fragment thereof are introduced into the T cell to generate the double-strand break.
  • the end-processing enzyme exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5'exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
  • the one or more proinflammatory cytokines are selected from the group consisting of: IFNy, IL-4, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and ⁇ - ⁇ .
  • a method of treating a cancer in a subject comprising, administering a CAR T cell or a composition contemplated herein to the subject.
  • a therapeutically effective amount of CAR T cells comprising a modified TCRa allele that is administered to the subject to clear the tumor is less that the therapeutically effective amount of CAR T cells lacking a modified TCRa that is administered to the subject to clear the tumor.
  • the cancer is a solid cancer.
  • the cancer is a liquid cancer.
  • the hematological malignancy is multiple myeloma.
  • the hematological malignancy is CLL.
  • a method of treating a B cell related condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of CAR T cells or a therapeutically effective amount of a composition contemplated herein to the subject.
  • the B cell related condition is multiple myeloma, non-
  • Hodgkin's lymphoma B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenia purpura, anti- phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly- arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti- phospholipid syndrome, ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy- chain disease, primary or immunocyte-associated amyloidosis, or monoclonal gammopathy of undetermined significance.
  • the B cell related condition is a B cell malignancy.
  • the B cell related condition is a plasma cell malignancy.
  • the B cell related condition is an autoimmune disease.
  • a method of increasing the efficacy of a CAR T cell comprising decreasing the availability of TCRa signaling components on the CAR T cell surface by introducing an engineered nuclease that creates a double-strand break (DSB) at a target site in a TCRa allele to modify the TCRa allele.
  • DSB double-strand break
  • the CAR T cell comprises one or more proviral integrants encoding the CAR and wherein the DSB is repaired by non-homologous end joining (HEJ).
  • HEJ non-homologous end joining
  • the CAR T cell comprises an RNA polymerase II promoter operably linked to a nucleic acid encoding a CAR incorporated into the TCRa allele by homology directed repair at the DSB.
  • the engineered nuclease is selected from the group consisting of: a meganuclease, a megaTAL, a TALEN, a ZFN, or a CRISPR/Cas nuclease.
  • the meganuclease is engineered from an I-Onul LHE.
  • the engineered nuclease is a megaTAL comprising a TALE
  • DNA binding domain and an engineered meganuclease.
  • the double-strand break is generated by an engineered endonuclease and an end-processing enzyme.
  • an mRNA encoding the engineered endonuclease and an mRNA encoding the end-processing enzyme are introduced into the T cell to generate the double-strand break.
  • an mRNA encoding the engineered endonuclease, a viral-self cleaving peptide, and an end-processing enzyme are introduced into the T cell to generate the double-strand break.
  • an mRNA encoding the engineered endonuclease, an IRES element, and an end-processing enzyme are introduced into the T cell to generate the double- strand break.
  • an mRNA encoding the engineered endonuclease fused to an end-processing enzyme or biologically active fragment thereof is introduced into the T cell to generate the double-strand break.
  • the end-processing enzyme exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
  • the CAR T cells bound to a target antigen secrete an increased amount of one or more proinflammatory cytokines selected from the group consisting of: IFNy, IL-4, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and ⁇ ⁇ , compared to a CAR T cell bound to the target antigen that lacks a modified TCRa allele.
  • proinflammatory cytokines selected from the group consisting of: IFNy, IL-4, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and ⁇ ⁇
  • Figure 1 shows the VCNs at day 10, for two representative donors for anti-BCMA CAR T cells cultured using the standard culture process (no EP), cells electroporated but received no mRNA (EP only), and cells electroporated with mRNA encoding a megaTAL that targets the TCRa gene (TCRa KO), both at large scale and small scale.
  • Figure 2 shows representative growth curves over 10 days, for anti-BCMA CAR T cells cultured using the standard culture process (no EP), cells electroporated but received no mRNA (EP only), and cells electroporated with mRNA encoding a megaTAL that targets the TCRa gene (TCRa KO), both at large scale and small scale.
  • FIG 3 shows representative FACS plots for two donors for anti-BCMA CAR expression and TCRa disruption.
  • Anti-BCMA CAR expression was determined by staining cells with BCMA antigen conjugated to PE fluorophore.
  • TCRa disruption was determined by anti-CD3 staining.
  • Figure 4 shows a graphical representation of the FACS data shown in Figure 3.
  • Figure 5A shows representative data for antigen dependent IFNy release for anti- BCMA CAR T cells cultured using the standard culture process (no EP), cells electroporated but received no mRNA (EP only), and cells electroporated with mRNA encoding a megaTAL that targets the TCRa gene (TCRa KO) cultured in the presence of BCMA positive and BCMA negative cell lines.
  • Figure 5B shows representative data for antigen dependent release of IL-4, IL-10, TNFa, IL-8, IL-5, IL-6, GM-CSF, and MIP- ⁇ for anti-BCMA CAR T cells cultured using the standard culture process (no EP), and cells electroporated with mRNA encoding a megaTAL that targets the TCRa gene (TCRa KO) +/- Trex2 cultured in the presence of BCMA positive and BCMA negative cell lines.
  • Figure 6A shows the in vivo efficacy of anti-BCMA CAR T cells manufactured using the standard process (no EP), EP only, and TCRa KO treatments in a Daudi xenograft model up to day 27.
  • Figure 6B shows the in vivo efficacy of anti-BCMA CAR T cells manufactured using the standard process (no EP), EP only, and TCRa KO treatments in a Daudi xenograft model up to day 43.
  • Figure 7 shows a representative FACS plot of anti-CD19 CAR expression in T cells transduced with lentivirus encoding an anti-CD 19 CAR (LV-T cells) and T cells where an anti- CD 19 CAR has been inserted into the TCRa gene using homology directed repair (HR-T cells).
  • Figure 8A shows the in vitro cytotoxicity of anti-CD 19 CAR T cells (LV-T cells) and anti-CD 19 CAR T cells (LV-T cells) prepared from two donors in a co-culture assay with CD19 expressing Nalm-6 cells at an E:T ratio of 1 : 1.
  • Figure 8B shows representative data for antigen dependent IFNy release for untransduced control T cells and anti-CD 19 CAR T cells (LV-T cells) and anti-CD 19 CAR T cells (LV-T cells) prepared from two donors after 24 hours of co-culture with CD 19 expressing Nalm-6 cells at an E:T ratio of 1 : 1.
  • Figure 9 shows a representative FACS plot of anti-CD19 CAR expression in T cells transduced with lentivirus encoding an anti-CD 19 CAR and T cells where an anti-CD 19 CAR has been inserted into the TCRa gene using homology directed repair.
  • Figure 10A shows the in vitro cytotoxicity of anti-CD19 CAR T cells (LV-T cells) and anti-CD19 CAR T cells (LV-T cells) prepared from two donors in a co-culture assay with CD19 expressing Nalm-6 cells at an E:T ratio of 1 : 1.
  • Figure 10B shows representative data for antigen dependent IFNy, IL-2, IL-4, and TNFa release for anti-CD19 CAR T cells (LV-T cells) and anti-CD19 CAR T cells (LV-T cells) prepared from two donors after 24 hours of co-culture with CD 19 expressing Nalm-6 cells at an E:T ratio of 1 : 1.
  • Figure 11A shows the expression of T cell exhaustion markers for anti-CD 19 CAR T cells produced by lentiviral transduction (LV-CAR T cells) or homologous recombination HR- CAR T cells) cultured in the presence of CD 19 expressing Nalm-6 cells for 24 hours.
  • Figure 11B shows the expression of T cell exhaustion markers for anti-CD 19 CAR T cells produced by lentiviral transduction (LV-CAR T cells) or homologous recombination HR- CAR T cells) cultured in the presence of CD 19 expressing Nalm-6 cells for 72 hours.
  • SEQ ID NO: 1 sets forth the polynucleotide sequence of I-Onul.
  • SEQ ID NO: 2 sets forth the polypeptide sequence encoded by SEQ ID NO: 1.
  • SEQ ID NOs: 3 and 4 set forth illustrative examples of TCRa target sites for genome editing.
  • SEQ ID Nos: 5-7 set forth polypeptide sequences of engineered I-Onul variants.
  • SEQ ID NO: 8 sets forth the polypeptide sequence of an anti-CD 19 CAR.
  • SEQ ID NO: 9 sets forth the polynucleotide sequence of plasmid, pBW1021.
  • SEQ ID NO: 10 sets forth the TCRa I-Onul megaTAL target site.
  • SEQ ID NO: 11 sets forth the polypeptide sequence of an illustrative example of a TCRa I-Onul megaTAL.
  • SEQ ID NO: 12 sets forth the polypeptide sequence of an illustrative example of an anti-BCMA CAR.
  • SEQ ID NO: 13 sets forth the polynucleotide sequence of plasmid, pBW400.
  • SEQ ID NOs: 14-24 set forth the amino acid sequences of various linkers.
  • SEQ ID NOs: 25-49 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.
  • the invention generally relates, in part, to improved immune effector cell compositions and methods of manufacturing the same.
  • the immune effector cells contemplated in particular embodiments comprise a chimeric antigen receptor (CAR) and a precise disruption or modification in one or more T cell receptor loci, which leads to disruption of TCR expression and signaling.
  • CAR chimeric antigen receptor
  • genome edited CAR T cells comprising one or more modified TCRa alleles manufactured by the methods contemplated herein are imbued with increased therapeutic efficacy, e.g., increased antigen dependent secretion of proinflammatory cytokines, e.g., ⁇ , TNFa, IL-2, and other molecules including cytotoxins, e.g., Perforin, Granzyme A, Granzyme B, that increase the cytolytic activity of CAR T cells toward tumor cells.
  • proinflammatory cytokines e.g., ⁇ , TNFa, IL-2
  • cytotoxins e.g., Perforin, Granzyme A, Granzyme B
  • CAR T cells comprise a modification of one or more alleles of the T cell receptor alpha (TCRa) gene.
  • modification of one or more TCRa alleles ablates or substantially ablates expression of the TCRa allele(s), decreases expression of the TCRa allele(s), and/or impairs, substantially impairs, or ablates one or more functions of the TCRa allele(s) or renders the TCRa allele(s) non-functional.
  • TCRa functions include, but are not limited to, recruiting CD3 to the cell surface, MHC dependent recognition and binding of antigen, activation of TCRaP signaling.
  • HEJ of the ends of the cleaved genomic sequence may result in a cell with normal TCR expression, expression of a loss-of- or gain-of-function TCR, or preferably, a cell that lacks functional TCR expression, e.g., lacks the ability to recruit CD3 to cell surface, activate TCRaP signaling, recognize and bind MHC -antigen complexes.
  • the repair is biased toward NHEJ and loss-of-function by using engineered endonucleases and an end-processing enzyme, e.g., 3' to 5' exonuclease (Trex2) or biologically active fragment thereof.
  • a TCRa allele is repaired with the sequence of the template by homologous recombination at the DNA break-site.
  • the repair template comprises a polynucleotide sequence that encodes a CAR.
  • compositions contemplated herein represent a quantum improvement compared to existing adoptive cell therapies.
  • an element means one element or one or more elements.
  • the term "about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
  • the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
  • an “immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • effector functions e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC.
  • immune effector cells include natural killer (NK) cells.
  • immune effector cells include natural killer T (NKT) cells.
  • the immune effector cell is a CAR T cell.
  • T cell or "T lymphocyte” are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T cell can be a helper T cell (HTL; CD4 + T cell) CD4 + T cell, a cytotoxic T cell (CTL; CD8 + T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8 + T cell), CD4 + CD8 + T cell, CD4 CD8 " T cell, or any other subset of T cells.
  • the T cell is an NKT cell.
  • Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. In preferred
  • T cells are modified to express a CAR.
  • Protent T cells and “young T cells,” are used interchangeably in particular embodiments and refer to T cell phenotypes wherein the T cell is capable of proliferation and a concomitant decrease in differentiation.
  • the young T cell has the phenotype of a "naive T cell.”
  • young T cells comprise one or more of, or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38.
  • young T cells comprise one or more of, or all of the following biological markers: CD62L, CD127, CD197, and CD38.
  • the young T cells lack expression of CD57, CD244, CD160, PD-1, CTLA4, ⁇ 3, and LAG3.
  • proliferation refers to an increase in cell division, either symmetric or asymmetric division of cells.
  • proliferation refers to the symmetric or asymmetric division of T cells.
  • Increased proliferation occurs when there is an increase in the number of cells in a treated sample compared to cells in a non-treated sample.
  • differentiated T cells acquire immune effector cell functions.
  • T cell manufacturing or “methods of manufacturing T cells' or comparable terms refer to the process of producing a therapeutic composition of T cells, which manufacturing methods may comprise one or more of, or all of the following steps: harvesting, stimulation, activation, genome editing, and expansion.
  • ex vivo refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions.
  • "ex vivo" procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances.
  • tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be "in vitro " though in certain embodiments, this term can be used interchangeably with ex vivo.
  • in vivo refers generally to activities that take place inside an organism, such as cell self-renewal and cell proliferation or expansion.
  • in vivo expansion refers to the ability of a cell population to increase in number in vivo.
  • cells are engineered or modified in vivo.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule ⁇ e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event including, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • a "stimulatory molecule,” refers to a molecule on a T cell that specifically binds with a cognate stimulatory ligand.
  • a “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell ⁇ e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
  • an antigen presenting cell e.g., an aAPC, a dendritic cell, a B-cell, and the like
  • a cognate binding partner referred to herein as a "stimulatory molecule”
  • Stimulatory ligands include, but are not limited to CD3 ligands, e.g., an anti-CD3 antibody and CD2 ligands, e.g., anti-CD2 antibody, and peptides, e.g., CMV, HPV, EBV peptides.
  • activation refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In particular embodiments, activation can also be associated with induced cytokine production, and detectable effector functions.
  • activated T cells refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone are insufficient for full activation of the T cell and one or more secondary or costimulatory signals are also required. Thus, T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary costimulatory signals.
  • Co-stimulation can be evidenced by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the CD3/TCR complex or through CD2.
  • a "costimulatory signal,” refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation, cytokine production, and/or upregulation or downregulation of particular molecules (e.g., CD28).
  • costimulatory ligand refers to a molecule that binds a costimulatory molecule.
  • a costimulatory ligand may be soluble or provided on a surface.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand (e.g., anti-CD28 antibody).
  • Allogeneic refers to cells wherein the donor and recipient species are the same but the cells are genetically different.
  • “Syngeneic,” as used herein, refers to cells wherein the donor and recipient species are the same, the donor and recipient are different individuals, and the donor cells and recipient cells are genetically identical.
  • Xenogeneic refers to cells wherein the donor and recipient species are different.
  • the terms "individual” and “subject” are often used interchangeably and refer to any animal that exhibits a symptom of cancer or other immune disorder that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein.
  • Suitable subjects e.g., patients
  • laboratory animals such as mouse, rat, rabbit, or guinea pig
  • farm animals such as a cat or dog
  • domestic animals or pets such as a cat or dog.
  • Non-human primates and, preferably, human patients are included.
  • Typical subjects include human patients that have, have been diagnosed with, or are at risk or having, cancer or another immune disorder.
  • the term "patient” refers to a subject that has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer, autoimmune disease, immune disorder, etc. Treatment can optionally involve delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • preventing etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer, autoimmune disease, immune disorder, etc. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
  • the phrase "ameliorating at least one symptom of refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated, e.g., cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency.
  • the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).
  • the term “amount” refers to "an amount effective” or “an effective amount” of genome edited CAR T cells sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • prophylactically effective amount refers to an amount of genome edited CAR T cells effective to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the
  • prophylactically effective amount is less than the therapeutically effective amount.
  • a “therapeutically effective amount” of genome edited CAR T cells may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the genome edited CAR T cell to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to "treat" a subject (e.g., a patient).
  • compositions contemplated in particular embodiments, to be administered can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • immune disorder refers to a disease that evokes a response from the immune system.
  • immune disorder refers to a cancer, an autoimmune disease, or an immunodeficiency.
  • immune disorders encompasses infectious disease.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).
  • metastatic tumor refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • Benign or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • the amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.
  • autoimmune disease refers to a disease in which the body produces an
  • immunogenic i.e., immune system
  • the immune system loses its ability to recognize some tissue or system within the body as "self and targets and attacks it as if it were foreign.
  • Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g., hemolytic anemia and anti- immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g., systemic lupus erythematosus).
  • multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision.
  • Autoimmune diseases include, for instance, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
  • Immunodeficiency means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance.
  • Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.
  • infectious disease refers to a disease that can be transmitted from person to person or from organism to organism, and is caused by a microbial or viral agent (e.g., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • a microbial or viral agent e.g., common cold.
  • Infectious diseases include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • a composition contemplated herein e.g., genome edited CAR T cells comprising one or more modified TCRa alleles, to produce, elicit, or cause a greater response (i.e., physiological response) compared to the response caused by CAR T cells expressing the same or substantially the same CAR, but where the TCRa alleles have not been modified (control composition).
  • a measurable response may include an increase proinflammatory cytokine release, e.g., ⁇ , increases in cytolytic activity, or an increase in CAR expression, among other measurable responses apparent from the understanding in the art and the description herein.
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by a vehicle or control composition.
  • composition contemplated herein refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser response (i.e., physiological response) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable response may include a decrease in endogenous TCR expression or function, and the like.
  • a “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by a vehicle or control composition.
  • maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in a cell, as compared to the response caused by either vehicle, a control molecule/composition, or the response in a particular cell lineage.
  • a comparable response is one that is not significantly different or measurable different from the reference response.
  • binding affinity or “specifically binds” or “specifically bound” or “specific binding” or “specifically targets” as used herein, describe binding of one molecule to another at greater binding affinity than background binding.
  • a binding domain “specifically binds” to a target molecule if it binds to or associates with a target molecule with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 10 5 M "1 .
  • a binding domain binds to a target with a K a greater than or equal to about 10 6 M “1 , 10 7 M “1 , 10 8 M “1 , 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , 10 12 M “1 , or 10 13 M “1 .
  • "High affinity” binding domains refers to those binding domains with a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , at least 10 13 M “1 , or greater.
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 "5 M to 10 "13 M, or less).
  • Kd equilibrium dissociation constant
  • Affinities of binding domain polypeptides contemplated in particular embodiments can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assays using labeled ligands, or using a surface-plasmon resonance device such as the Biacore T100, which is available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC system or EnSpire that are available from Corning and Perkin Elmer respectively (see also, e.g.,
  • the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
  • an “antigen (Ag)” refers to a compound, composition, or substance, e.g., lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.
  • a “target antigen” or “target antigen or interest” is an antigen that a binding domain of an engineered antigen receptor contemplated herein, is designed to bind.
  • the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • An “epitope” or “antigenic determinant” refers to the region of an antigen to which a binding agent binds.
  • isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally -occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • isolated polynucleotide also refers to a complementary DNA (cDNA), a recombinant DNA, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
  • isolated protein refers to in vitro synthesis, isolation, and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
  • isolated cell refers to a non-naturally occurring cell, e.g., a T cell that does not exist in nature, a modified T cell, an engineered T cell, etc., that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
  • Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination.
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • HDR homology-directed repair
  • This process requires nucleotide sequence homology, uses a "donor” molecule as a template to repair a "target” molecule ⁇ i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis- dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule.
  • Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, polypeptides contemplated herein are used for targeted double-stranded DNA cleavage.
  • a “target site” or “target sequence” is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist.
  • exogenous molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods.
  • exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • lipid-mediated transfer i.e., liposomes, including neutral and cationic lipids
  • electroporation direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, or other organelle, or a naturally-occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, endogenous TCRs.
  • a “gene,” refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
  • a gene includes, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • genomic editing refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell's genome, which restores, corrects, and/or modifies expression of a gene. Genome editing contemplated in particular
  • embodiments comprises introducing one or more engineered nucleases into a cell to generate DNA lesions at a target site in the cell's genome, optionally in the presence of a donor repair template.
  • genetically engineered or “genetically modified” refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or non- targeted to a particular site in a cell's genome. In one embodiment, genetic modification is site specific. In one embodiment, genetic modification is not site specific.
  • the genome edited cells manufactured by the methods contemplated in particular embodiments comprise improved CAR T cell compositions.
  • a CAR T cell comprises one or more modified TCRa alleles.
  • the inventors have discovered that genome edited CAR T cells comprising one or more modified TCRa alleles manufactured by the methods contemplated herein are imbued with increased therapeutic efficacy, e.g., increased antigen dependent secretion of proinflammatory cytokines, e.g., IFNy, TNFa, IL-2, and other molecules including cytotoxins, e.g., Perforin, Granzyme A, Granzyme B, that increase the cytolytic activity of CAR T cells toward tumor cells.
  • proinflammatory cytokines e.g., IFNy, TNFa, IL-2
  • cytotoxins e.g., Perforin, Granzyme A, Granzyme B
  • increased CAR T cell efficacy is due, in part, to decreased TCR ⁇ receptor expression freeing up accessory signaling components for CARs, increased cytokine secretion, and reduction of tonic signaling.
  • genome edited CAR T cells comprise one or more TCRa alleles modified by the gene editing compositions and methods contemplated herein.
  • genome edited CAR T cells comprise one or more modified TCRa alleles; and a nucleic acid encoding a CAR.
  • genome edited CAR T cells comprise one or more modified TCRa alleles; and one or more proviral integrants comprising a nucleic acid encoding a CAR.
  • the proviral integrants are retroviral- or lentiviral -based proviral integrants.
  • genome edited CAR T cells comprise one or more modified TCRa alleles; wherein the one or more TCRa alleles have been modified by inserting a donor repair template comprising a nucleic acid encoding a CAR into the one or more TCRa alleles by homology directed repair.
  • a method of making a CAR T cell comprising one or more modified TCRa alleles comprises activating a population of T cells and stimulating the population of T cells to proliferate; introducing an engineered nuclease into the population of T cells to generate a double-strand break at a TCRa allele; and introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cell.
  • CAR chimeric antigen receptor
  • a method of making a CAR T cell comprising one or more modified TCRa alleles comprises activating a population of T cells and stimulating the population of T cells to proliferate; and transducing the T cell with a viral vector encoding a CAR.
  • Expression of the engineered nuclease creates a double-strand break at a target site in the TCRa allele that is preferentially repaired by HEJ.
  • an engineered endonuclease is introduced into the cell with a 3 ' end-processing enzyme, e.g., a 3 ' to 5 ' exonuclease such as Trex2, to increase the bias to repair the double-strand break by NHEJ.
  • the T cell is transduced with a retroviral vector, e.g., gamma retroviral vector or lentiviral vector, encoding a CAR. In one embodiment, the T cell is transduced with a lentiviral vector encoding a CAR.
  • a retroviral vector e.g., gamma retroviral vector or lentiviral vector
  • the T cell is transduced with a lentiviral vector encoding a CAR.
  • a method of making a CAR T cell comprising one or more modified TCRa alleles comprises activating a population of T cells and stimulating the population of T cells to proliferate; and transducing the T cell with a viral vector comprising a donor repair template that comprises a nucleic acid encoding a CAR; wherein expression of the engineered nuclease creates a double-strand break at a target site in the TCRa allele, and the donor repair template is incorporated into the TCRa allele by homology directed repair (HDR) at the site of the double-strand break (DSB).
  • the donor template further comprises an RNApol II promoter operably linked to a nucleic acid encoding a CAR.
  • Genome edited CAR T cells contemplated in particular embodiments may be autologous/autogeneic ("self) or non-autologous ("non-self,” e.g., allogeneic, syngeneic or xenogeneic).
  • autologous refers to cells from the same subject.
  • the T cells are obtained from a mammalian subject. In a more preferred embodiment, the T cells are obtained from a primate subject. In the most preferred embodiment, the T cells are obtained from a human subject.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation.
  • an isolated or purified population of T cells is used.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • a specific subpopulation of T cells, expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques.
  • a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38 is further isolated by positive or negative selection techniques.
  • the manufactured T cell compositions do not express or do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, ⁇ 3, and LAG3.
  • an isolated or purified population of T cells expresses one or more of the markers including, but not limited to a CD3 + , CD4 + , CD8 + , or a combination thereof
  • the T cells are isolated from an individual and first activated and stimulated to proliferate in vitro prior to undergoing genome editing.
  • T cells are often subjected to one or more rounds of stimulation, activation and/or expansion.
  • T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;
  • T cells are activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of the genome editing compositions into the T cells.
  • T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of the genome editing compositions into the T cells.
  • T cells are activated at the same time that genome editing compositions are introduced into the T cells.
  • a costimulatory ligand is presented on an antigen presenting cell ⁇ e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate
  • costimulatory molecule on a T cell thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex, mediates a desired T cell response.
  • Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor, and a ligand that specifically binds with B7-H3.
  • a costimulatory ligand comprises an antibody or antigen binding fragment thereof that specifically binds to a costimulatory molecule present on a T cell, including but not limited to, CD27, CD28, 4- IBB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD7, LIGHT, KG2C, B7-H3, and a ligand that specifically binds with CD83.
  • a costimulatory ligand comprises an antibody or antigen binding fragment thereof that specifically binds to a costimulatory molecule present on a T cell, including but not limited to, CD27, CD28, 4- IBB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD7, LIGHT, KG2C, B7-H3, and a ligand that specifically binds with CD83.
  • LFA-1 lymphocyte function-associated antigen- 1
  • Suitable costimulatory ligands further include target antigens, which may be provided in soluble form or expressed on APCs or aAPCs that bind engineered antigen receptors expressed on genome edited T cells.
  • a method of generating a genome edited CAR T cell comprises activating a population of cells comprising T cells and expanding the population of T cells.
  • T cell activation can be accomplished by providing a primary stimulation signal through the T cell TCR/CD3 complex and by providing a secondary costimulation signal through an accessory molecule, e.g., CD28.
  • the TCR/CD3 complex may be stimulated by contacting the T cell with a suitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonal antibody.
  • a suitable CD3 binding agent e.g., a CD3 ligand or an anti-CD3 monoclonal antibody.
  • CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.
  • induction of T cell responses requires a second, costimulatory signal.
  • a CD28 binding agent can be used to provide a costimulatory signal.
  • CD28 binding agents include but are not limited to: natural CD 28 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7 family of proteins, such as B7-1(CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibody or fragment thereof capable of crosslinking the CD28 molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are coupled to the same surface.
  • binding agents that provide stimulatory and costimulatory signals are localized on the surface of a cell. This can be accomplished by transfecting or transducing a cell with a nucleic acid encoding the binding agent in a form suitable for its expression on the cell surface or alternatively by coupling a binding agent to the cell surface.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are displayed on antigen presenting cells.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are provided on separate surfaces.
  • one of the binding agents that provides stimulatory and costimulatory signals is soluble (provided in solution) and the other agent(s) is provided on one or more surfaces.
  • binding agents that provide stimulatory and
  • costimulatory signals are both provided in a soluble form (provided in solution).
  • the methods for making genome edited CAR T cells contemplated herein comprise activating T cells with anti-CD3 and anti-CD28 antibodies.
  • expanding T cells activated by the methods contemplated herein further comprises culturing a population of cells comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or any hourly integer value in between.
  • the T cell composition may be cultured for 14 days.
  • T cells are cultured for about 21 days.
  • the T cell compositions are cultured for about 2-3 days. Several cycles of stimulation/activation/expansion may also be desired such that culture time of T cells can be 60 days or more.
  • conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives suitable for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN- ⁇
  • IL-4 interleukin-7
  • IL-21 e.g., GM-CSF
  • IL-10 interleukin-12
  • IL-15 e.g., IL-15
  • TGFp transforming growth factor
  • cell culture media include, but are not limited to RPMI 1640, Clicks, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02).
  • PBMCs or isolated T cells are contacted with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, and/or IL-15.
  • a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
  • cytokines such as IL-2, IL-7, and/or IL-15.
  • artificial APC made by engineering K562, U937, 721.221, T2, and C1R cells to direct the stable expression and secretion, of a variety of costimulatory molecules and cytokines.
  • K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the AAPC cell surface.
  • Populations of T cells can be expanded by aAPCs expressing a variety of costimulatory molecules including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86.
  • the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells.
  • aAPCs provided in WO 03/057171 and US2003/0147869 are hereby incorporated by reference in their entirety.
  • a method for generating a CAR T cell comprising a modified TCRa allele comprises introducing one or more engineered nucleases contemplated herein into the population of T cells to generate a double-strand break in a TCRa allele.
  • the one or more nucleases contemplated herein are introduced into the T cell prior to activation and stimulation. In another embodiment, the one or more nucleases contemplated herein are introduced into the T cell at about the same time that the T cell is stimulated.
  • the one or more nucleases contemplated herein are introduced into the T cell after the T cell activation and stimulation, e.g., about 1, 2, 3, or 4 days after.
  • the nucleases introduced into the T cells include, but are not limited to an endonuclease, e.g., a meganuclease, a megaTAL, a TALEN, a ZFN, or a CRISPR/Cas nuclease; and optionally an end-processing nuclease or biologically active fragment thereof, e.g., 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the endonuclease and end-processing nuclease may be expressed as a fusion protein
  • the one or more nucleases are introduced into a T cell using a vector.
  • the one or more nucleases are preferably introduced into a T cell as mRNAs.
  • the nucleases may be introduced into the T cells by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and the like.
  • Genome editing methods contemplated in particular embodiments comprise introducing one or more engineered nucleases contemplated herein into a population of activated and stimulated T cells in order to create a DSB at a target site that is preferentially repaired via NHEJ, and subsequently transducing the population of T cells with a vector encoding a CAR.
  • Genome editing methods contemplated in particular embodiments comprise introducing one or more engineered nucleases contemplated herein into a population of activated and stimulated T cells in order to create a DSB at a target site and optionally, subsequently introducing one or more donor repair templates into the population of T cells that will be incorporated into the cell's genome at the DSB site by homologous recombination.
  • one or more donor templates comprising a polynucleotide encoding a CAR are introduced into the population of T cells.
  • the donor templates may be introduced into the T cells by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and the like.
  • the one or more nucleases are introduced into the T cell by mRNA electroporation and the one or more donor repair templates are introduced into the T cell by viral transduction.
  • the one or more nucleases are introduced into the T cell by mRNA electroporation and the one or more donor repair templates are introduced into the T cell by AAV transduction.
  • the AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10.
  • the AAV vector may comprise ITRs from AAV2 and a serotype from AAV6.
  • the donor repair template comprises one or more homology arms.
  • the term "homology arms" refers to a nucleic acid sequence in a donor template that is identical, or nearly identical, to the DNA sequence flanking the DNA break introduced by the nuclease at a target site.
  • the donor template comprises a 5 ' homology arm that comprises a nucleic acid that is identical or nearly identical to the DNA sequence 5 ' of the DNA break site.
  • the donor template comprises a 3 ' homology arm that comprises a nucleic acid that is identical or nearly identical to the DNA sequence 3 ' of the DNA break site.
  • the donor template comprises a 5 ' homology arm and a 3 ' homology arm.
  • suitable lengths of homology arms may be independently selected, and include but are not limited to: about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, about 2500 bp, about 2600 bp, about 2700 bp, about 2800 bp, about 2900 bp, or about 3000 bp, or longer homology arms, including all intervening lengths of homology arms.
  • suitable homology arm lengths include, but are not limited to: about 100 bp to about 3000 bp, about 200 bp to about 3000 bp, about 300 bp to about 3000 bp, about 400 bp to about 3000 bp, about 500 bp to about 3000 bp, about 500 bp to about 2500 bp, about 500 bp to about 2000 bp, about 750 bp to about 2000 bp, about 750 bp to about 1500 bp, or about 1000 bp to about 1500 bp, including all intervening lengths of homology arms.
  • the lengths of the 5 ' and 3 ' homology arms are
  • 5 ' homology arm is about 1500 bp and the 3 ' homology arm is about 1000 bp. In one embodiment, the 5 ' homology arm is about 600 bp and the 3 ' homology arm is about 600 bp.
  • Donor repair templates may further comprises one or more polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, contemplated elsewhere herein.
  • polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleot
  • the donor repair template comprises a 5 ' homology arm, an
  • RNA polymerase II promoter a CAR, and a 3 ' homology arm.
  • the one or more nucleases are introduced into the T cell by mRNA electroporation and the one or more donor repair templates are introduced into the T cell by lentiviral transduction.
  • the lentiviral vector backbone may be derived from HTV- 1, HIV-2, visna-maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), or simian immunodeficiency virus (SIV).
  • the lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV).
  • IDLV vectors comprising an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) are preferred.
  • the one or more donor repair templates may be delivered prior to, simultaneously with, or after the one or more engineered nucleases are introduced into a cell.
  • the one or more donor repair templates are delivered simultaneously with the one or more engineered nucleases.
  • the one or more donor repair templates are delivered prior to the one or more engineered nucleases, for example, seconds to hours to days before the one or more donor repair templates, including, but not limited to about 1 min. to about 30 min., about 1 min. to about 60 min., about 1 min. to about 90 min., about 1 hour to about 24 hours before the one or more engineered nucleases or more than 24 hours before the one or more engineered nucleases.
  • the one or more donor repair templates are delivered after the nuclease, preferably within about 1, 2, 3, 4, 5, 6, 7, or 8 hours; more preferably, within about 1, 2, 3, or 4 hours; or more preferably, within about 4 hours.
  • the one or more donor repair templates may be delivered using the same delivery systems as the one or more engineered nucleases.
  • the donor repair templates and engineered nucleases may be encoded by the same vector, e.g., an IDLV lentiviral vector or an AAV vector (e.g., AAV6).
  • the engineered nuclease(s) are delivered by mRNA electroporation and the donor repair templates are delivered by transduction with an AAV vector.
  • methods of generating CAR T cells with one or more modified TCRa alleles comprises contacting the cells with a stimulatory agent and
  • costimulatory agent such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, and/or IL-15 and/or one or more agents that modulate a PI3K cell signaling pathway.
  • PI3K inhibitor refers to a nucleic acid, peptide, compound, or small organic molecule that binds to and inhibits at least one activity of PI3K.
  • the PI3K proteins can be divided into three classes, class 1 PDKs, class 2 PDKs, and class 3 PDKs.
  • Class 1 PI3Ks exist as heterodimers consisting of one of four pi 10 catalytic subunits (pi 10a, pi 10 ⁇ , pi 105, and pi 10 ⁇ ) and one of two families of regulatory subunits.
  • a PI3K inhibitor targets the class 1 PI3K inhibitors.
  • a PI3K inhibitor will display selectivity for one or more isoforms of the class 1 PI3K inhibitors (i.e., selectivity for pi 10a, pi 10 ⁇ , pi 105, and pi 10 ⁇ or one or more of pi 10a, pi 10 ⁇ , pi 10 ⁇ , and pi 10 ⁇ ).
  • a PI3K inhibitor will not display isoform selectivity and be considered a "pan-PI3K inhibitor.”
  • a PI3K inhibitor will compete for binding with ATP to the PI3K catalytic domain.
  • PI3K inhibitors suitable for use particular embodiments include, but are not limited to, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (class 1 PI3K inhibitor; pi 10a, pi 10 ⁇ , and pi 10 ⁇ isoforms, Oncothyreon).
  • selective PI3K inhibitors include, but are not limited to
  • pan-PI3K inhibitors include, but are not limited to BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.
  • the PI3K inhibitor is ZSTK474. D. NUCLEASES
  • Immune effector cell compositions contemplated in particular embodiments are generated by genome editing accomplished with engineered nucleases targeting one or more TCRa alleles.
  • CAR T cells are edited using engineered nucleases, designed to bind and cleave a target DNA sequence in the T cell receptor alpha (TCRa) gene.
  • the engineered nucleases contemplated in particular embodiments can be used to introduce a double-strand break in a target polynucleotide sequence, which may be repaired by nonhomologous end joining (HEJ) in the absence of a polynucleotide template, e.g., a donor repair template, and subsequently transduced with a virus encoding a CAR; or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template encoding a CAR.
  • HEJ nonhomologous end joining
  • HDR homology directed repair
  • Engineered nucleases contemplated in certain embodiments can also be engineered as nickases, which generate single-stranded DNA breaks that can be repaired using the cell's base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template.
  • BER base-excision-repair
  • NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function.
  • An “engineered nuclease” refers to a nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and/or modified to bind a DNA binding target sequence adjacent to a DNA cleavage target sequence.
  • the engineered nuclease may be designed and/or modified from a naturally occurring nuclease or from a previously engineered nuclease.
  • Engineered nucleases contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • additional functional domains e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • engineered HEs are introduced into a T cell with an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the HE and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self- cleaving peptide or an IRES element.
  • homing endonucleases meganucleases
  • megaTALs transcription activator-like effector nucleases
  • ZFNs zinc finger nucleases
  • CRISPR clustered regularly-interspaced short palindromic repeats
  • the nucleases contemplated herein comprise one or more heterologous DNA-binding and cleavage domains (e.g., ZFNs, TALENs, megaTALs), (Boissel et al., 2014; Christian etal., 2010).
  • the DNA-binding domain of a naturally-occurring nuclease may be altered to bind to a selected target site (e.g., a
  • meganuclease that has been engineered to bind to site different than the cognate binding site).
  • meganucleases have been designed to bind target sites different from their cognate binding sites (Boissel etal., 2014).
  • a nuclease requires a nucleic acid sequence to target the nuclease to a target site (e.g., CRISPR/Cas).
  • a homing endonuclease or meganuclease is engineered to bind to, and to introduce single-stranded nicks or double-strand breaks (DSBs) in a TCRa locus.
  • DSBs double-strand breaks
  • LAGLIDADG LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E)XK.
  • Engineered HEs do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. Engineered HEs may be obtained by making one or more amino acid alterations, e.g., mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring HE or previously engineered HE. In particular embodiments, an engineered HE comprises one or more amino acid alterations to the DNA recognition interface.
  • a "DNA recognition interface” refers to the HE amino acid residues that interact with nucleic acid target bases as well as those residues that are adjacent.
  • the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to recognize a particular nucleic acid target sequence.
  • the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies significantly and is a feature of any natural or engineered HE.
  • an engineered HE contemplated in particular embodiments may be derived by constructing libraries of HE variants in which one or more amino acid residues localized in the DNA recognition interface of the natural HE (or a previously engineered HE) are varied.
  • the libraries may be screened for target cleavage activity against each predicted TCRa locus target sites using cleavage assays (see e.g., Jarjour et al, 2009. Nuc. Acids Res. 37(20): 6871-6880).
  • LAGLIDADG homing endonucleases are the most well studied family of meganucleases, are primarily encoded in archaea and in organellar DNA in green algae and fungi, and display the highest overall DNA recognition specificity. LHEs comprise one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single chain monomers, respectively. Structural studies of LAGLIDADG proteins identified a highly conserved core structure (Stoddard 2005), characterized by an ⁇ fold, with the
  • LAGLIDADG motif belonging to the first helix of this fold belongs to the first helix of this fold.
  • the highly efficient and specific cleavage of LHE' s represent a protein scaffold to derive novel, highly specific endonucleases.
  • engineering LHEs to bind and cleave a non-natural or non-canonical target site requires selection of the appropriate LHE scaffold, examination of the target locus, selection of putative target sites, and extensive alteration of the LHE to alter its DNA contact points and cleavage specificity, at up to two-thirds of the base-pair positions in a target site.
  • LHEs from which engineered LHEs may be designed include, but are not limited to I-AabMI, I-AaeMI, I- Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I- CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I- Gpil, I-GzeMI, I-GzeMII, I-GzeMHI, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I- Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMII, I-OsoMIII, I-O
  • the engineered LHE is selected from the group consisting of: I- CpaMI, I-Hj eMI, I-Onul, I-PanMI, and SmaMI.
  • the engineered LHE is I-Onul. See e.g., SEQ ID NOs: 1 and 2.
  • engineered I-Onul LHEs targeting the human TCRa gene were generated from a natural I-Onul. In a preferred embodiment, engineered I-Onul LHEs targeting the human TCRa gene were generated from a previously engineered I-Onul. In one embodiment, engineered I-Onul LHEs were generated against a human TCRa gene target site set forth in SEQ ID NO: 3. In one embodiment, engineered I-Onul LHEs were generated against a human TCRa gene target site set forth in SEQ ID NO: 4.
  • the engineered I-Onul LHE comprises one or more amino acid substitutions in the DNA recognition interface.
  • the I-Onul LHE comprises at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%), at least 97%, at least 98%, or at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U. S. A. 2011 Aug 9; 108(32): 13077-13082) or an engineered variant of I-Onul as
  • the I-Onul LHE comprises at least 70%, more preferably at least 80%), more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identity with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U. S. A . 2011 Aug 9; 108(32): 13077-13082) or an engineered variant of I-Onul as set forth in SEQ ID NOs: 5, 6, or 7, or further engineered variants thereof.
  • an engineered I-Onul LHE comprises one or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68 to 82, 180 to 203 and 223 to 240 of I-Onul (SEQ ID NO: 2) or an engineered variant of I-Onul as set forth in SEQ ID NOs: 5, 6, or 7, or further engineered variants thereof.
  • an engineered I-Onul LHE comprises one or more amino acid substitutions or modifications at additional positions situated anywhere within the entire I-Onul sequence.
  • the residues which may be substituted and/or modified include but are not limited to amino acids that contact the nucleic acid target or that interact with the nucleic acid backbone or with the nucleotide bases, directly or via a water molecule.
  • an engineered I-Onul LHE contemplated herein comprises one or more substitutions and/or modifications, preferably at least 5, preferably at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 25 in at least one position selected from the position group consisting of positions: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240 of I-Onul (SEQ ID NO: 2) or an engineered variant of I-Onul as set forth in SEQ ID NOs: 5, 6, or 7, or further engineered variants thereof.
  • an engineered I-Onul LHE contemplated herein comprises one or more amino acids substitutions and/or modifications selected from the group consisting of: L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, N75R, S78M, K80R, L138M, S159P, E178D, C180Y, F182G, I186K, S188V, S190G, K191N, L192A, G193K, Q195Y, Q197G, V199R, T203S, K207R, Y223S, K225W, and D236E.
  • the I-Onul LHE has an amino acid sequence as set forth in SEQ ID NOs: 5, 6, or 7, or further engineered variants thereof.
  • megaTAL nuclease that binds to and cleaves a target region of a TCRa locus.
  • a "megaTAL” refers to an engineered nuclease comprising an engineered TALE DNA binding domain and an engineered meganuclease, and optionally comprise one or more linkers and/or additional functional domains, e.g., an end- processing enzymatic domain of an end-processing enzyme that exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template- independent DNA polymerases activity.
  • end-processing enzyme e.g., an end- processing enzymatic domain of an end-processing enzyme that exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5'exonuclease (e.g.
  • a megaTAL can be introduced into a T cell with an end-processing enzyme that exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template- independent DNA polymerases activity.
  • the megaTAL and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
  • TALE DNA binding domain is the DNA binding portion of transcription activator-like effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome (see e.g., Kay etal., 2007. Science 318:648-651).
  • TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, e.g., AvrBs3 from Xanthomonas campestris pv.
  • vesicatoria Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and
  • TALE proteins for deriving and designing DNA binding domains are disclosed in U.S. Patent No. 9,017,967, and references cited therein, all of which are incorporated herein by reference in their entireties.
  • a megaTAL comprises a TALE DNA binding domain comprising one or more repeat units that are involved in binding of the TALE DNA binding domain to its corresponding target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length.
  • Each TALE DNA binding domain repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di-Residue (RVD), typically at positions 12 and/or 13 of the repeat.
  • RVD Repeat Variable Di-Residue
  • the natural (canonical) code for DNA recognition of these TALE DNA binding domains has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T.
  • C cytosine
  • NG binds to T
  • NI to A NI to A
  • NN binds to G or A
  • NG binds to T.
  • non-canonical (atypical) RVDs are contemplated.
  • Illustrative examples of non-canonical RVDs suitable for use in particular megaTALs contemplated in particular embodiments include, but are not limited to HH, KH, H, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G); NI, KI, RI, HI, SI for recognition of adenine (A); NG, HG, KG, RG for recognition of thymine (T); RD, SD, HD, ND, KD, YG for recognition of cytosine (C); NV, HN for recognition of A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at position 13 is absent.
  • Additional illustrative examples of RVDs suitable for use in particular megaTALs contemplated in particular embodiments further include those disclosed in U. S. Patent No. 8,614,
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units.
  • a megaTAL comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TALE DNA binding domain repeat units.
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5-13 repeat units, more preferably 7-12 repeat units, more preferably 9-11 repeat units, and more preferably 9, 10, or 11 repeat units.
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3 to 30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of a set of TALE repeat units, i.e., an additional C-terminal half-TALE DNA binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed elsewhere herein, infra).
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3.5 to 30.5 repeat units.
  • a megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5,
  • a megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5-13.5 repeat units, more preferably 7.5-12.5 repeat units, more preferably 9.5-11.5 repeat units, and more preferably 9.5, 10.5 , or 11.5 repeat units .
  • a megaTAL comprises an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains/units, a "C-terminal domain (CTD)” polypeptide, and an engineered meganuclease.
  • NTD N-terminal domain
  • the NTD sequence if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA.
  • the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA binding domain (0 is amino acid 1 of the most N-terminal repeat unit).
  • the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA binding domain.
  • a megaTAL contemplated herein comprises an NTD polypeptide of at least about amino acids +1 to +122 to at least about +1 to +137 of aXanthomonas TALE protein (0 is amino acid 1 of the most N- terminal repeat unit).
  • the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of aXanthomonas TALE protein.
  • a megaTAL contemplated herein comprises an NTD polypeptide of at least amino acids +1 to +121 of aRalstonia TALE protein (0 is amino acid 1 of the most N-terminal repeat unit).
  • the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Ralstonia TALE protein.
  • CTD C-terminal domain
  • the CTD sequence if present, may be of any length as long as the TALE DNA binding domain repeat units retain the ability to bind DNA.
  • the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA binding domain (the first 20 amino acids are the half-repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 443, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids C-terminal to the last full repeat of the TALE DNA binding domain.
  • a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 is amino acid 1 of a half- repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Xanthomonas TALE protein.
  • a megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Ralstonia TALE protein (-20 is amino acid 1 of a half-repeat unit C-terminal to the last C-terminal full repeat unit).
  • the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Ralstonia TALE protein.
  • a megaTAL contemplated herein comprises a fusion polypeptide comprising a TALE DNA binding domain engineered to bind a target sequence, a meganuclease engineered to bind and cleave a target sequence, and optionally an NTD and/or CTD polypeptide, optionally joined to each other with one or more linker polypeptides contemplated elsewhere herein.
  • a megaTAL comprising TALE DNA binding domain, and optionally an NTD and/or CTD polypeptide is fused to a linker polypeptide which is further fused to an engineered meganuclease.
  • the TALE DNA binding domain binds a DNA target sequence that is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA binding domain of the meganuclease.
  • the megaTALs contemplated herein increase the specificity and efficiency of genome editing.
  • a megaTAL contemplated herein comprises one or more TALE DNA binding repeat units and an engineered LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I- CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-Gpil, I-GzeMI, I- GzeMn, I-GzeMin, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-Ncrll, I-Ncrl, I- NcrMI, I-OheMI, I-Onul, I-OsoMI, I-OsoMI, I-Os
  • a megaTAL contemplated herein comprises an NTD, one or more TALE DNA binding repeat units, a CTD, and an engineered LHE selected from the group consisting of: I-AabMI, I-AaeMI, I- Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I- CpaMI, I-CpaMII, I-CpaMni, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I- Gpil, I-GzeMI, I-GzeMII, I-GzeMHI, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I- Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-
  • a megaTAL contemplated herein comprises an NTD, about 9.5 to about 11.5 TALE DNA binding repeat units, and an engineered I-Onul LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, I- CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-Ej eMI, I- GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMin, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I- MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-On
  • a megaTAL contemplated herein comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, or about 11.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an engineered I-Onul LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-Ej eMI, I- GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I-GzeMin, I-HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I- MveMI, I-N
  • TALEN that binds to and cleaves a target region of a TCRa locus.
  • a "TALEN” refers to an engineered nuclease comprising an engineered TALE DNA binding domain contemplated elsewhere herein and an endonuclease domain (or endonuclease half-domain thereof), and optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end- processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • end-processing enzymatic domain of an end- processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonucle
  • a TALEN can be introduced into a T cell with an end- processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the TALEN and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
  • targeted double-stranded cleavage is achieved with two TALENs, each comprising an endonuclease half-domain that can be used to reconstitute a catalytically active cleavage domain.
  • targeted double-stranded cleavage is achieved using a single polypeptide comprising a TALE DNA binding domain and two endonuclease half-domains.
  • TALENs contemplated in particular embodiments comprise an NTD, a TALE DNA binding domain comprising about 3 to 30 repeat units, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeat units, and an endonuclease domain or half-domain.
  • TALENs contemplated in particular embodiments comprise an NTD, a TALE DNA binding domain comprising about 3.5 to 30.5 repeat units, e.g., about 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 repeat units, a CTD, and an endonuclease domain or half- domain.
  • TALENs contemplated in particular embodiments comprise an NTD of about 121 amino acids to about 137 amino acids as disclosed elsewhere herein, a TALE DNA binding domain comprising about 9.5 to about 11.5 repeat units (i.e., about 9.5, about 10.5, or about 11.5 repeat units), a CTD of about 20 amino acids to about 85 amino acids, and an
  • a TALEN comprises an endonuclease domain of a type restriction endonuclease.
  • Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
  • Certain restriction enzymes e.g., Type-IIS
  • TALENs comprise the endonuclease domain (or endonuclease half-domain) from at least one Type-IIS restriction enzyme and one or more TALE DNA-binding domains contemplated elsewhere herein.
  • Type-IIS restriction endonuclease domains suitable for use in TALENs contemplated in particular embodiments include endonuclease domains of the at least 1633 Type-IIS restriction endonucleases disclosed at "rebase.neb.com/cgi-bin/sublist?S.”
  • Type-IIS restriction endonuclease domains suitable for use in TALENs contemplated in particular embodiments include those of endonucleases selected from the group consisting of: Aar I, Ace III, Aci I, Alo I, Alw26 I, Bae I, Bbr7 I, Bbv I, Bbv ⁇ , BbvC I, Bcc I, Bce83 I, Bee A I, Beef I, Beg I, BciV I, Bfi I, Bin I, Bmg I, BpulO I, BsaX I, Bsb I, BscA I, BscG I, BseR I, BseY I, Bsi I, Bsm I, BsmA I, BsmF I, Bsp24 I, BspG I, BspM I, BspNC I, Bsr I, BsrB I, BsrD I, BstF5 I, Btr I
  • a TALEN contemplated herein comprises an endonuclease domain of the Fok I Type-IIS restriction endonuclease.
  • a TALEN contemplated herein comprises a TALE DNA binding domain and an endonuclease half-domain from at least one Type-IIS restriction endonuclease to enhance cleavage specificity, optionally wherein the endonuclease half-domain comprises one or more amino acid substitutions or modifications that minimize or prevent
  • cleavage half-domains suitable for use in particular embodiments contemplated in particular embodiments include those disclosed in U.S. Patent Publication Nos. 20050064474; 20060188987, 20080131962, 20090311787; 20090305346; 20110014616, and 20110201055, each of which are incorporated by reference herein in its entirety.
  • a zinc finger nuclease that binds to and cleaves a target site in a TCRa locus.
  • ZFN refers to an engineered nuclease comprising one or more zinc finger DNA binding domains and an endonuclease domain (or endonuclease half-domain thereof), and optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonucle
  • a ZFN can be introduced into a T cell with an end- processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease (e.g., Trex2), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
  • the ZFN and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
  • targeted double-stranded cleavage is achieved using two ZFNs, each comprising an endonuclease half-domain can be used to reconstitute a catalytically active cleavage domain.
  • targeted double-stranded cleavage is achieved with a single polypeptide comprising one or more zinc finger DNA binding domains and two endonuclease half-domains.
  • a ZNF comprises a TALE DNA binding domain contemplated elsewhere herein, a zinc finger DNA binding domain, and an endonuclease domain (or endonuclease half-domain) contemplated elsewhere herein.
  • a ZNF comprises a zinc finger DNA binding domain, and a meganuclease contemplated elsewhere herein.
  • the ZFN comprises a zinger finger DNA binding domain that has one, two, three, four, five, six, seven, or eight or more zinger finger motifs and an endonuclease domain (or endonuclease half-domain).
  • a single zinc finger motif is about 30 amino acids in length.
  • Zinc fingers motifs include both canonical C2H2 zinc fingers, and non-canonical zinc fingers such as, for example, C3H zinc fingers and C 4 zinc fingers.
  • Zinc finger binding domains can be engineered to bind any DNA sequence.
  • Candidate zinc finger DNA binding domains for a given 3 bp DNA target sequence have been identified and modular assembly strategies have been devised for linking a plurality of the domains into a multi -finger peptide targeted to the corresponding composite DNA target sequence.
  • Other suitable methods known in the art can also be used to design and construct nucleic acids encoding zinc finger DNA binding domains, e.g., phage display, random mutagenesis, combinatorial libraries, computer/rational design, affinity selection, PCR, cloning from cDNA or genomic libraries, synthetic construction and the like. (See, e.g., U.S. Pat. No.
  • Individual zinc finger motifs bind to a three or four nucleotide sequence.
  • the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc finger motifs in an engineered zinc finger binding domain. For example, for ZFNs in which the zinc finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger binding domain; a nine-nucleotide target sequence is bound by a three-finger binding domain, etc.
  • DNA binding sites for individual zinc fingers motifs in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the linker sequences between the zinc finger motifs in a multi-finger binding domain.
  • ZNFs contemplated herein comprise, a zinc finger DNA binding domain comprising two, three, four, five, six, seven or eight or more zinc finger motifs, and an endonuclease domain or half-domain from at least one Type-IIS restriction enzyme and one or more TALE DNA-binding domains contemplated elsewhere herein.
  • ZNFs contemplated herein comprise, a zinc finger DNA binding domain comprising three, four, five, six, seven or eight or more zinc finger motifs, and an endonuclease domain or half-domain from at least one Type-IIS restriction enzyme selected from the group consisting of: Aar I, Ace ⁇ , Aci I, Alo I, Alw26 I, Bae I, Bbr7 I, Bbv I, Bbv II, BbvC I, Bcc I, Bce83 I, BceA I, Beef I, Beg I, BciV I, Bfi I, Bin I, Bmg I, BpulO I, BsaX I, Bsb I, BscA I, BscG I, BseR I, BseY I, Bsi I, Bsm I, BsmA I, BsmF I, Bsp24 I, BspG I, BspM I, BspNC I, Bsr I, Bs
  • ZNFs contemplated herein comprise, a zinc finger DNA binding domain comprising three, four, five, six, seven or eight or more zinc finger motifs, and an endonuclease domain or half-domain from the Fok I Type-IIS restriction endonuclease.
  • a ZFN contemplated herein comprises a zinc finger DNA binding domain and an endonuclease half-domain from at least one Type-IIS restriction endonuclease to enhance cleavage specificity, optionally wherein the endonuclease half-domain comprises one or more amino acid substitutions or modifications that minimize or prevent
  • a CRISPR Clustered Regularly Interspaced Short
  • Palindromic Repeats)/Cas (CRISPR Associated) nuclease system is engineered to bind to, and to introduce single-stranded nicks or double-strand breaks (DSBs) in, one or more TCRa loci.
  • the CRISPR/Cas nuclease system is a recently engineered nuclease system based on a bacterial system that can be used for mammalian genome engineering. See, e.g., Jinek et al. (2012) Science 337:816-821; Cong etal. (2013) Science 339:819-823; Mali et al. (2013) Science 339:823-826; Qi et al.
  • the CRISPR/Cas nuclease system comprises Cas nuclease and one or more RNAs that recruit the Cas nuclease to the target site, e.g., a transactivating cRNA (tracrRNA) and a CRISPR RNA (crRNA), or a single guide RNA (sgRNA).
  • crRNA and tracrRNA can engineered into one polynucleotide sequence referred to herein as a "single guide RNA" or "sgRNA.”
  • the Cas nuclease is engineered as a double-stranded DNA endonuclease or a nickase or catalytically dead Cas, and forms a target complex with a crRNA and a tracrRNA, or sgRNA, for site specific DNA recognition and site-specific cleavage of the protospacer target sequence located within the TCRa locus.
  • the protospacer motif abuts a short protospacer adjacent motif (PAM), which plays a role in recruiting a Cas/RNA complex.
  • Cas polypeptides recognize PAM motifs specific to the Cas polypeptide.
  • the CRISPR/Cas system can be used to target and cleave either or both strands of a double- stranded polynucleotide sequence flanked by particular 3' PAM sequences specific to a particular Cas polypeptide.
  • PAMs may be identified using bioinformatics or using
  • the Cas nuclease comprises one or more heterologous DNA binding domains, e.g., a TALE DNA binding domain or zinc finger DNA binding domain. Fusion of the Cas nuclease to TALE or zinc finger DNA binding domains increases the DNA cleavage efficiency and specificity.
  • a Cas nuclease optionally comprises one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease ⁇ e.g., Trex2), 5' flap endonuclease, helicase or template- independent DNA polymerases activity.
  • additional functional domains e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 -3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease ⁇ e.g., Trex2), 5' flap endonuclease, helicase or template- independent DNA polymerases activity.
  • a Cas nuclease can be introduced into a T cell with an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5'exonuclease ⁇ e.g., Trex2), 5' flap endonuclease, helicase or template- independent DNA polymerases activity.
  • the Cas nuclease and 3 ' processing enzyme may be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
  • the Cas nuclease is Cas9 or Cpfl.
  • Cas9 polypeptides suitable for use in particular embodiments contemplated in particular embodiments may be obtained from bacterial species including, but not limited to: Enterococcus faecium, Enterococcus italicus, Listeria innocua, Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus equinus,
  • Streptococcus gallolyticus Streptococcus macacae, Streptococcus mutans, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus gordonii, Streptococcus infantarius, Streptococcus macedonicus, Streptococcus mitis, Streptococcus pasteurianus, Streptococcus suis, Streptococcus vestibularis, Streptococcus sanguinis, Streptococcus downei, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria subflava, Lactobacillus brevis,
  • Lactobacillus buchneri Lactobacillus casei, Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus johnsonii,
  • Lactobacillus rhamnosus Lactobacillus ruminis, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Corynebacterium accolens, Corynebacterium diphtheriae, Corynebacterium matruchotii, Campylobacter jejuni, Clostridium perfringens, Treponema vincentii, Treponema phagedenis, and Treponema denticola.
  • Cpfl polypeptides suitable for use in particular embodiments contemplated in particular embodiments may be obtained from bacterial species including, but not limited to: Francisella spp., Acidaminococcus spp., Prevotella spp., Lachnospiraceae spp., among others.
  • conserveed regions of Cas9 orthologs include a central HNH endonuclease domain and a split RuvC/RNase H domain.
  • Cpfl orthologs possess a RuvC/RNase H domain but no discemable HNH domain.
  • the HNH and RuvC-like domains are each responsible for cleaving one strand of the double-stranded DNA target sequence.
  • the HNH domain of the Cas9 nuclease polypeptide cleaves the DNA strand complementary to the tracrRNAxrRNA or sgRNA.
  • the RuvC-like domain of the Cas9 nuclease cleaves the DNA strand that is not- complementary to the tracrRNAxrRNA or sgRNA.
  • Cpfl is predicted to act as a dimer wherein each RuvC-like domain of Cpfl cleaves either the complementary or non- complementary strand of the target site.
  • a Cas9 nuclease variant e.g., Cas9 nickase
  • Cas9 nickase comprising one or more amino acids additions, deletions, mutations, or substitutions in the HNH or RuvC-like endonuclease domains that decreases or eliminates the nuclease activity of the variant domain.
  • Cas9 HNH mutations that decrease or eliminate the nuclease activity in the domain include, but are not limited to: S. pyogenes (D10A); S. thermophilis (D9A); T. denticola (D13A); and N. meningitidis (D16A).
  • Cas9 RuvC-like domain mutations that decrease or eliminate the nuclease activity in the domain include, but are not limited to: S. pyogenes (D839A, H840A, or N863A); S. thermophilis (D598A, H599A, or N622A); T. denticola (D878A, H879A, orN902A); and N. meningitidis (D587A, H588A, or N611A).
  • S. pyogenes D839A, H840A, or N863A
  • S. thermophilis D598A, H599A, or N622A
  • T. denticola D878A, H879A, orN902A
  • N. meningitidis D587A, H588A, or N611A.
  • T cells comprising one or more modified TCRa alleles are engineered to express one or more chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • T cells are engineered by introducing a D SB in one or more TCRa alleles and by subsequently transducing the T cells with a vector encoding a CAR.
  • T cells are engineered by introducing a D SB in one or more TCRa alleles in the presence of a donor repair template encoding a CAR.
  • a CAR is inserted at a DSB in a single TCRa allele.
  • the engineered T cells contemplated herein comprise a CAR that is not inserted at a TCRa allele.
  • the genome edited T cells express CARs that redirect cytotoxicity toward tumor cells.
  • CARs are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with a T cell receptor-activating
  • chimeric describes being composed of parts of different proteins or DNAs from different origins.
  • a CAR comprises an extracellular domain that binds to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and an intracellular signaling domain.
  • a specific target antigen also referred to as a binding domain or antigen-specific binding domain
  • the main characteristic of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors.
  • MHC major histocompatibility
  • CARs comprise an extracellular binding domain that specifically binds to a target polypeptide, e.g., a target antigen, expressed on tumor cell.
  • a target polypeptide e.g., a target antigen
  • binding domain the terms, "binding domain,” "extracellular domain,” “extracellular binding domain,” “antigen binding domain,” “antigen-specific binding domain,” and “extracellular antigen specific binding domain,” are used interchangeably and provide a CAR with the ability to specifically bind to the target antigen of interest.
  • a binding domain may comprise any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or component thereof).
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof.
  • an “antibody” refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • a target antigen such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • Antibodies include antigen binding fragments, e.g., Camel Ig (a camelid antibody or VHH fragment thereof), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domain antibody (sdAb, Nanobody) or other antibody fragments thereof.
  • Camel Ig a camelid antibody or VHH fragment thereof
  • Ig NAR Ig NAR
  • Fab fragments fragments
  • Fab' fragments fragments
  • F(ab)'2 fragments F(ab)'3 fragments
  • Fv single chain Fv antibody
  • scFv single chain Fv antibody
  • dsFv disulfide stabilized Fv protein
  • the term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies) and antigen binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
  • the binding domain is an scFv.
  • the binding domain is a camelid antibody.
  • the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor, 5T4, ⁇ ⁇ ⁇ 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvni, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRoc, GD2, GD3, Glypican-3 (GPC3), HL A- A 1 +M AGE 1 , HLA-A2+MAGE1, HL A- A3 +M AGE 1 , HLA-Al+NY-ESO-1, HL A-A2+NY-ESO- 1 , H
  • the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72.
  • the CARs comprise an extracellular binding domain, e.g., antibody or antigen binding fragment thereof that binds an antigen, wherein the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • an extracellular binding domain e.g., antibody or antigen binding fragment thereof that binds an antigen
  • the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • the CARs comprise linker residues between the various domains.
  • a "variable region linking sequence,” is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
  • a linker separates one or more heavy or light chain variable domains, hinge domains,
  • CARs comprise one, two, three, four, or five or more linkers.
  • the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.
  • the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long.
  • the binding domain of the CAR is followed by one or more
  • spacer domains refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419).
  • the spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3.
  • the spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • the spacer domain comprises the CH2 and CH3 of IgGl, IgG4, or IgD.
  • the binding domain of the CAR is linked to one or more "hinge domains," which plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation.
  • a CAR generally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM).
  • the hinge domain may be derived either from a natural, synthetic, semisynthetic, or recombinant source.
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be altered.
  • the hinge domain comprises a CD8a hinge region.
  • the hinge is a PD-1 hinge or CD152 hinge.
  • the "transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell.
  • the TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • Illustrative TM domains may be derived from (i.e., comprise at least the
  • transmembrane region(s) of the alpha or beta chain of the T-cell receptor CD35, CD3s, CD3y, CD3C, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, and PD-1.
  • a CAR comprises a TM domain derived from CD8a.
  • a CAR contemplated herein comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TM domain and the intracellular signaling domain of the CAR.
  • a glycine-serine linker provides a particularly suitable linker.
  • a CAR comprises an intracellular signaling domain.
  • An "intracellular signaling domain,” refers to the part of a CAR that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal.
  • intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal.
  • T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
  • a CAR comprises an intracellular signaling domain that comprises one or more "costimulatory signaling domains" and a "primary signaling domain.”
  • Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • Illustrative examples of IT AM containing primary signaling domains suitable for use in CARs contemplated in particular embodiments include those derived from FcRy, FcR- ⁇ , CD3y, CD35, CD38, CD3 ⁇ CD22, CD79a, CD79b, and CD66d.
  • a CAR comprises a CD3 ⁇ primary signaling domain and one or more costimulatory signaling domains.
  • the intracellular primary signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • a CAR comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing CAR receptors.
  • costimulatory signaling domain refers to an intracellular signaling domain of a costimulatory molecule.
  • a CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3 ⁇ primary signaling domain.
  • the CAR comprises: an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8a, CD154, and PD-1; one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3s, CD3C, CD22, CD79a, CD79b, and CD66d.
  • an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72
  • a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8a, CD154, and PD-1
  • polypeptides are contemplated herein, including, but not limited to, CARs, meganucleases, megaTALs, TALENs, ZFNs, and Cas nucleases, fusion polypeptides, and vectors that express polypeptides.
  • a "polypeptide” includes fusion
  • Polypeptides and other variants.
  • Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the
  • polypeptide for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • isolated peptide or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
  • polypeptides contemplated in particular embodiments include, but are not limited to meganucleases, megaTALs, TALENs, ZFNs, Cas nucleases, end- processing nucleases, therapeutic polypeptides and fusion polypeptides and variants thereof.
  • Polypeptide variants include biologically active "polypeptide fragments.”
  • biologically active fragment or “minimal biologically active fragment” refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%>, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
  • Polypeptide fragments refer to a polypeptide, which can be monomelic or multimeric that has an amino-terminal deletion, a carboxyl- terminal deletion, and/or an internal deletion or substitution of one or more amino acids of a naturally-occurring or recombinantly-produced polypeptide.
  • a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long.
  • polypeptide fragments include DNA binding domains, nuclease domains, antibody fragments, extracellular ligand binding domains, signaling domains, transmembrane domains, multimerization domains, and the like.
  • Polypeptides include "polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a polypeptide by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide.
  • polypeptides include polypeptides having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367- 382), U.S. Pat. No. 4,873,192, Watson, J. D.
  • a variant will contain one or more conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments, polypeptides include polypeptides having at least about and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non- polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • Suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson etal. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).
  • Polypeptide variants further include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties ⁇ e.g., pegylated molecules).
  • Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art.
  • Variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect functional activity of the proteins are also variants.
  • the polynucleotide sequences encoding them can be separated by and IRES sequence as disclosed elsewhere herein.
  • Fusion polypeptides contemplated in particular embodiments include fusion polypeptides.
  • fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
  • Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
  • two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
  • a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and/or self-cleaving polypeptides.
  • a fusion protein contemplated herein comprises one or more exodomains, extracellular ligand binding domains, or antigen binding domain, a
  • transmembrane domain and or one or more intracellular signaling domains, and optionally one or more multimerization domains.
  • polypeptides include polypeptides having at least about include, but are not limited to:
  • Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C- terminus.
  • the polypeptides of the fusion protein can be in any order.
  • Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques.
  • Fusion polypeptides may optionally comprise a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
  • a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
  • Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al, Gene 40:39-46, 1985; Murphy et al, Proc. Natl Acad. Sci.
  • Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
  • Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
  • Exemplary linkers include, but are not limited to the following amino acid sequences: glycine polymers (G) n ; glycine-serine polymers (Gi-5Si-5)n, where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; GGG (SEQ ID NO: 14); DGGGS (SEQ ID NO: 15); TGEKP (SEQ ID NO: 16) (see e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 17) (Pomerantz etal.
  • GGG SEQ ID NO: 14
  • DGGGS SEQ ID NO: 15
  • TGEKP SEQ ID NO: 16
  • KESGSVSSEQLAQFRSLD (SEQ ID NO: 20) (Bird et al, 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 21); LRQRDGERP (SEQ ID NO: 22); LRQKDGGGSERP (SEQ ID NO: 23); LRQKD(GGGS) 2 ERP (SEQ ID NO: 24).
  • flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91 : 11099- 11103 (1994) or by phage display methods.
  • Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template.
  • a polypeptide cleavage site can be put into any linker peptide sequence.
  • Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).
  • Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al., 1997. J Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
  • Exemplary protease cleavage sites include, but are not limited to the cleavage sites of poty virus NIa proteases (e.g., tobacco etch virus protease), poty virus HC proteases, poty virus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
  • poty virus NIa proteases e.g., tobacco etch virus protease
  • poty virus HC proteases e.
  • TEV tobacco etch virus protease cleavage sites
  • EXXYXQ(G/S) SEQ ID NO: 25
  • ENLYFQG SEQ ID NO: 26
  • ENLYFQS SEQ ID NO: 27
  • the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al, 2001. J. Gen. Virol. 82: 1027-1041).
  • the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
  • the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna vims (TaV) 2A peptide, a porcine teschovims-1 (PTV-1) 2A peptide, a Theilovims 2A peptide, and an encephalomyocarditis vims 2A peptide.
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A virus
  • TaV Thosea asigna vims
  • PTV-1 porcine teschovims-1
  • Theilovims 2A peptide a Theilovims 2A peptide
  • an encephalomyocarditis vims 2A peptide an encephalomyocarditis vims 2A peptide.
  • Exemplary 2A sites include the following sequences:
  • polynucleotides encoding a polypeptide or fusion polypeptide contemplated herein are provided.
  • polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double-stranded.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • ribozymes synthetic RNA
  • genomic RNA gRNA
  • RNA(+) plus strand RNA
  • RNA(-) minus strand RNA
  • crRNA single guide RNA
  • Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths.
  • polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • polynucleotides contemplated herein include, but are not limited to polynucleotides encoding meganucleases, megaTALs, TALENs, ZFNs, Cas nucleases, end-processing nucleases, CARs, therapeutic polypeptides, and polynucleotides comprising expression vectors, viral vectors, and transfer plasmids.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
  • a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions.
  • stringent conditions describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by -amino acid basis over a window of comparison.
  • a "percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg,
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • BESTFIT Pearson FASTA
  • FASTA Altschul et al.
  • TFASTA Pearson's Alignment of Altschul et al.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in
  • isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally -occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • isolated polynucleotide refers to a complementary DNA
  • cDNA a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
  • Terms that describe the orientation of polynucleotides include: 5' (normally the end of the polynucleotide having a free phosphate group) and 3' (normally the end of the
  • polynucleotide having a free hydroxyl (OH) group polynucleotide having a free hydroxyl (OH) group.
  • Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation.
  • the 5' to 3' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA].
  • pre-mRNA pre-messenger
  • the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as "template,” “antisense,” “minus,” or “non-coding” strand.
  • the term “reverse orientation” refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation.
  • complementarity refers to polynucleotides ⁇ i.e., a sequence of nucleotides) related by the base-pairing rules.
  • the complementary strand of the DNA sequence 5 * A G T C A T G 3' is 3' T C A G T A C 5'.
  • the latter sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'.
  • a sequence that is equal to its reverse complement is said to be a palindromic sequence.
  • Complementarity can be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there can be “complete” or “total” complementarity between the nucleic acids.
  • nucleic acid cassette refers to genetic sequences within the vector which can express one or more RNAs.
  • the nucleic acid cassette contains one or more polynucleotide(s)-of-interest.
  • the nucleic acid cassette contains one or more expression control sequences operably linked to one or more polynucleotide(s)-of-interest.
  • Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes.
  • the cassette can be removed from or inserted into other polynucleotide sequences, e.g., a plasmid or viral vector, as a single unit.
  • Polynucleotides include polynucleotide(s)-of-interest. As used herein, the term
  • polynucleotide-of-interest refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein.
  • polynucleotide sequences that may encode a polypeptide, or fragment of variant thereof, as contemplated herein.
  • polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection.
  • polynucleotides comprising particular allelic sequences are provided. Alleles are endogenous polynucleotide sequences that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides.
  • a polynucleotide-of-interest comprises a donor repair template encoding a CAR.
  • a polynucleotide-of-interest comprises an inhibitory polynucleotide including, but not limited to, a crRNA, a tracrRNA, a single guide RNA (sgRNA), an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
  • an inhibitory polynucleotide including, but not limited to, a crRNA, a tracrRNA, a single guide RNA (sgRNA), an siRNA, an miRNA, an shRNA, a ribozyme or another inhibitory RNA.
  • RNA short interfering RNA
  • siRNA short polynucleotide sequence that mediates a process of sequence-specific post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetic RNAi in animals (Zamore et al, 2000, Cell, 101, 25-33; Fire et al, 1998, Nature, 391, 806; Hamilton et al, 1999, Science, 286, 950-951; Lin et al, 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886).
  • miRNA or “microRNA” s refer to small non-coding RNAs of 20-22 nucleotides, typically excised from -70 nucleotide fold-back RNA precursor structures known as pre-miRNAs. miRNAs negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target.
  • shRNA or short hairpin RNA refer to double-stranded structure that is formed by a single self-complementary RNA strand.
  • ribozyme refers to a catalytically active RNA molecule capable of site-specific cleavage of target mRNA.
  • a donor repair template comprises an inhibitory RNA comprises one or more regulatory sequences, such as, for example, a strong constitutive pol ⁇ , e.g., human or mouse U6 snRNA promoter, the human and mouse HI RNA promoter, or the human tRNA-val promoter, or a strong constitutive pol ⁇ promoter, as described elsewhere herein.
  • polynucleotides contemplated in particular embodiments may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons,
  • promoters and/or enhancers such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons,
  • transcriptional termination signals and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated in particular embodiments that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
  • vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
  • vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC)
  • bacteriophages such as lambda phage or M13 phage
  • animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • retrovirus including lentivirus
  • adenovirus e.g., adeno-associated virus
  • herpesvirus e.g., herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g., SV40
  • expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V 5-DESTTM, pLenti6/V 5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • the term "episomal” refers to a vector that is able to replicate without integration into host's chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • the vector is engineered to harbor the sequence coding for the origin of DNA replication or "ori" from an alpha, beta, or gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast.
  • the host cell comprises the viral replication
  • Alpha herpesviruses have a relatively short reproductive cycle, variable host range, efficiently destroy infected cells and establish latent infections primarily in sensory ganglia.
  • alpha herpes viruses include HSV 1, HSV 2, and VZV.
  • Beta herpesviruses have long reproductive cycles and a restricted host range. Infected cells often enlarge. Latency can be maintained in the white cells of the blood, kidneys, secretory glands and other tissues.
  • Illustrative examples of beta herpes viruses include CMV, HHV-6 and HHV-7.
  • Gamma-herpesviruses are specific for either T or B lymphocytes, and latency is often demonstrated in lymphoid tissue.
  • Illustrative examples of gamma herpes viruses include EBV and HHV-8.
  • “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector— origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters may be used.
  • a polynucleotide is a vector, including but not limited to expression vectors and viral vectors, and includes exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
  • An "endogenous control sequence” is one which is naturally linked with a given gene in the genome.
  • An “exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • a “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
  • An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
  • promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide- of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
  • a constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a "cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
  • Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and PI 1 promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein
  • CMV
  • HSP90B1 heat shock protein 70kDa
  • HSP70 heat shock protein 70kDa
  • ⁇ -kinesin ⁇ - ⁇
  • the human ROSA 26 locus Irions et al, Nature Biotechnology 25, 1477 - 1482 (2007)
  • Ubiquitin C promoter Ubiquitin C promoter
  • PGK phosphogly cerate kinase- 1
  • CAG cytomegalovirus enhancer/chicken ⁇ -actin
  • MND myeloproliferative sarcoma virus enhancer
  • a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
  • condition expression may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression;
  • conditional expression of a polynucleotide-of-interest e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
  • inducible promoters/sy stems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the "GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
  • steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the "GeneSwitch”
  • polynucleotides comprises at least one (typically two) site(s) for recombination mediated by a site specific recombinase.
  • recombinase or "site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten or more.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
  • Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, OC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.
  • the polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms "recombination sequence,"
  • recombination site or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.
  • loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)).
  • Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al.
  • Suitable recognition sites for the FLP recombinase include, but are not limited to: FRT (McLeod, et al, 1996), Fi,F 2 , F3 (Schlake and Bode, 1994), F 4 ,F 5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al, 1988), FRT(RE) (Senecoff et al, 1988).
  • recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme ⁇ Integrase, e.g., phi-c31.
  • the ⁇ C31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al, 2000).
  • attB and attP named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by ⁇ C31 homodimers (Groth et al, 2000).
  • the product sites, attL and attR, are effectively inert to further ⁇ > 1-mediated recombination (Belteki et al, 2003), making the reaction irreversible.
  • attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagaraj an et al. , 2001 ; Belteki et al. , 2003).
  • typical strategies position by homologous recombination an attP-bearing "docking site" into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion.
  • a polynucleotide contemplated herein comprises a repair template polynucleotide flanked by a pair of recombinase recognition sites.
  • the repair template polynucleotide is flanked by LoxP sites, FRT sites, or art sites.
  • polynucleotides contemplated herein include one or more polynucleotides-of-interest that encode one or more polypeptides.
  • the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.
  • an "internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al, 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
  • IRES immunoglobulin heavy-chain binding protein
  • VEGF vascular endothelial growth factor
  • FGF-2 fibroblast growth factor 2
  • IGFII insulinlike growth factor
  • EMCV encephelomycarditis virus
  • IRES VEGF IRES
  • Picornaviridae Dicistroviridae and Flaviviridae species
  • HCV Friend murine leukemia virus
  • MoMLV Moloney murine leukemia virus
  • the IRES used in polynucleotides contemplated herein is an EMCV IRES.
  • the polynucleotides comprise a consensus Kozak sequence.
  • Kozak sequence refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation.
  • the consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:50), where R is a purine (A or G) (Kozak, 1986. Cell 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).
  • vectors comprise a polyadenylation sequence 3 Of a polynucleotide encoding a polypeptide to be expressed.
  • polyA site or "polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase ⁇ .
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
  • polyA signals that can be used in a vector, includes an ideal polyA sequence (e.g., AATAAA, ATT AAA, AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a rabbit ⁇ -globin polyA sequence (rPgpA), or another suitable heterologous or endogenous poly A sequence known in the art.
  • ideal polyA sequence e.g., AATAAA, ATT AAA, AGTAAA
  • BGHpA bovine growth hormone polyA sequence
  • rPgpA rabbit ⁇ -globin polyA sequence
  • a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation.
  • the suicide gene is not immunogenic to the host harboring the polynucleotide or cell.
  • a certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
  • polynucleotides comprise gene segments that cause the genetically modified cells contemplated herein to be susceptible to negative selection in vivo.
  • Negative selection refers to an infused cell that can be eliminated as a result of a change in the in vivo condition of the individual.
  • the negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound.
  • Negative selection genes include, but are not limited to: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase.
  • HSV-I TK Herpes simplex virus type I thymidine kinase
  • HPRT hypoxanthine phosphribosyltransferase
  • APRT cellular adenine phosphoribosyl transferase
  • genetically modified cells comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro.
  • the positive selectable marker may be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting positive selection of cells carrying the gene.
  • Genes of this type are known in the art, and include, but are not limited to hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.
  • hph hygromycin-B phosphotransferase gene
  • DHFR dihydrofolate reductase
  • ADA adenosine deaminase gene
  • MDR multi-drug resistance
  • the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker.
  • the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other.
  • An example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT US91/08442 and PCT/US94/05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers.
  • Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt
  • preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.
  • Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker.
  • polynucleotides encoding one or more meganucleases, megaTALs, TALENs, ZFNs, Cas nucleases, end-processing nucleases, CARs, therapeutic polypeptides, and fusion polypeptides may be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods.
  • delivery of one or more polynucleotides encoding nucleases and/or donor repair templates may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
  • vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
  • non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.
  • Illustrative examples of non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial
  • chromosomes chromosomes, and viral vectors.
  • Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
  • polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
  • Lipofection reagents are sold commercially (e.g.,
  • Viral vectors comprising polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
  • viral vectors comprising engineered nucleases and/or donor repair templates are administered directly to an organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • embodiments contemplated in particular embodiments include, but are not limited to adeno- associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • herpes simplex virus adenovirus
  • vaccinia virus vectors for gene transfer include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
  • one or more polynucleotides encoding an engineered nuclease and/or donor repair template are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides.
  • an immune effector cell e.g., T cell
  • rAAV recombinant adeno-associated virus
  • AAV is a small (-26 nm) replication-defective, primarily episomal. non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • Recombinant AAV rAAV
  • rAAV are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • the ITR sequences are about 145 bp in length.
  • the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype.
  • a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2/AAV6.
  • the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6.
  • engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.
  • Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety.
  • one or more polynucleotides encoding a CAR, an engineered nuclease, or donor repair template are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising the one or more polynucleotides.
  • an immune effector cell e.g., T cell
  • a retrovirus e.g., lentivirus
  • retrovirus refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus
  • HaMuSV murine mammary tumor virus
  • MoMTV gibbon ape leukemia virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RS V Rous Sarcoma Virus
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include, but are not limited to: HIV (human
  • HIV based vector backbones i.e., HIV cis-acting sequence elements
  • HIV cis-acting sequence elements HIV based vector backbones
  • a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi ( ⁇ ) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
  • lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus.
  • integration defective lentivirus refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
  • HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41 A, K42A, H51 A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253
  • LTR long terminal repeat
  • FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
  • cPPT and CTS central polypurine tract and central termination sequences
  • Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal, 2000, Cell, 101 : 173.
  • packaging signal or "packaging sequence” refers to psi [ ⁇ ] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever etal, 1995. J of Virology, Vol. 69, No. 4; pp. 2101-2109.
  • RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • Examples of RNA export elements include, but are not limited to, the human
  • HIV immunodeficiency virus
  • RRE hepatitis B virus post- transcriptional regulatory element
  • expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et a/., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al, Mol. Cell. Biol, 5:3864); and the like (Liu et al, 1995, Genes Dev., 9: 1766).
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • HPRE hepatitis B virus
  • Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
  • Self-inactivating (SIN) vectors refers to replication-defective vectors, e.g., in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified ⁇ e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
  • heterologous promoters examples include, for example, viral simian virus 40 (SV40) ⁇ e.g., early or late), cytomegalovirus (CMV) ⁇ e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4 + presenting cells.
  • VSV-G vesicular stomatitis virus G-protein
  • lentiviral vectors are produced according to known methods. See e.g., Kutner et a/., BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et aZ Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
  • most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1.
  • a lentivirus e.g., HIV-1.
  • many different sources of retroviral and/or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein.
  • lentiviral vectors are known in the art, see Naldini et al, (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et al, 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein.
  • one or more polynucleotides encoding a CAR, an engineered nuclease, or donor repair template are introduced into an immune effector cell, by transducing the cell with an adenovirus comprising the one or more polynucleotides.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • adenovirus vectors which are replication deficient, may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991 ).
  • a unique helper cell line designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991 ).
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).
  • Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993).
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7: 1083-9 (1998)).
  • one or more polynucleotides encoding a CAR, an engineered nuclease, or donor repair template are introduced into an immune effector cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.
  • a herpes simplex virus e.g., HSV-1, HSV-2
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • the HSV based viral vector is deficient in one or more essential or non-essential HSV genes.
  • the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof.
  • HSV vectors are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb.
  • HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
  • compositions contemplated in particular embodiments may comprise one or more polypeptides, polynucleotides, vectors comprising same, and immune effector cell
  • compositions include, but are not limited to pharmaceutical compositions.
  • a "pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water
  • compositions comprise an amount genome edited CAR T cells manufactured by the methods contemplated herein.
  • the pharmaceutical compositions comprise CAR T cells comprising one or more modified and/or non-functional TCRa alleles and that express one or more CARs.
  • a pharmaceutical composition comprising the CAR T cells manufactured by the methods contemplated in particular embodiments may be administered at a dosage of about 10 2 to about 10 10 cells/kg body weight, about 10 5 to about 10 9 cells/kg body weight, about 10 5 to about 10 8 cells/kg body weight, about 10 5 to about 10 7 cells/kg body weight, about 10 7 to about 10 9 cells/kg body weight, or about 10 7 to about 10 8 cells/kg body weight, including all integer values within those ranges.
  • the number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.
  • the cells are generally in a volume of a liter or less, can be 500 mL or less, even 250 mL or 100 mL or less.
  • the density of the desired cells is typically greater than about 10 6 cells/mL and generally is greater than about 10 7 cells/mL, generally about 10 8 cells/mL or greater.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
  • T cells modified to express a CAR may be administered multiple times at dosages within these ranges.
  • the cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.
  • the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN- ⁇ , IL-2, IL-7, IL-15, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MTPla, etc.) as described herein to enhance engraftment and function of infused T cells.
  • mitogens e.g., PHA
  • lymphokines e.g., lymphokines, cytokines, and/or chemokines (e.g., IFN- ⁇ , IL-2, IL-7, IL-15, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MTPla, etc.) as described herein to enhance
  • compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • compositions comprising the modified T cells manufactured by the methods contemplated herein are used in the treatment of cancer.
  • the genome edited CAR T cells contemplated in particular embodiments may be administered either alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients, and/or with other components such as IL-2, IL-7, and/or IL-15 or other cytokines or cell populations.
  • pharmaceutical compositions contemplated herein comprise an amount of CAR T cells comprising one or more modified TCRa alleles, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions comprising genome edited CAR T cells contemplated in particular embodiments may further comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants;
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as antioxidants;
  • compositions contemplated in particular embodiments are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • the liquid pharmaceutical compositions may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents;
  • antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethyl enediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • the genome edited CAR T cell compositions are formulated in a pharmaceutically acceptable cell culture medium.
  • a pharmaceutically acceptable cell culture medium is a serum free medium.
  • Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
  • the serum-free medium is animal-free, and may optionally be protein-free.
  • the medium may contain biopharmaceutically acceptable recombinant proteins.
  • Animal-free medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources.
  • Protein-free in contrast, is defined as substantially free of protein.
  • Illustrative examples of serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X- vrvo 10.
  • compositions comprising genome edited CAR T cells contemplated herein are formulated in a solution comprising PlasmaLyte A.
  • compositions comprising genome edited CAR T cells contemplated herein are formulated in a solution comprising a cryopreservation medium.
  • cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
  • cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
  • compositions comprising genome edited CAR T cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
  • compositions contemplated herein comprise an effective amount of an expanded genome edited CAR T cell composition, alone or in combination with one or more therapeutic agents.
  • the CAR T cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy,
  • compositions may also be administered in combination with antibiotics.
  • therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer.
  • Exemplary therapeutic agents contemplated in particular embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, antiinflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.
  • compositions comprising CAR T cells contemplated herein may be administered in conjunction with any number of chemotherapeutic agents.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,
  • alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM)
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • tnetylenephosphoramide triethylenethiophosphaoramide and tnmethylolomelamine
  • nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard
  • nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine
  • antibiotics such as
  • aclacinomysins actinomycin, authramycin, azaserine, bleomycins, cactinomycin
  • calicheamicin carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
  • edatraxate defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
  • procarbazine PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®) and doxetaxel (TAXOTERE®); chlorambucil;
  • TAXOL® paclitaxel
  • TAXOTERE® doxetaxel
  • gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C;
  • aminopterin xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM (alitretinoin); ONTAKTM (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylomithine
  • retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM (alitretinoin); ONTAKTM (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the composition comprising CAR T cells is administered with an anti-inflammatory agent.
  • Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcino
  • exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and
  • CELEBREX® celecoxib
  • sialylates exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride.
  • Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
  • Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors.
  • the biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules.
  • Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporin ⁇ methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
  • Illustrative examples of therapeutic antibodies suitable for combination with the genome edited T cells contemplated in particular embodiments include but are not limited to, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab,
  • cixutumumab clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab,
  • ensituximab ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatum
  • racotumomab radretumab, nlotumumab, ntuximab, robatumumab, satumomab, sibrotuzumab, siltuximab, pumpuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab,
  • compositions contemplated herein are administered in conjunction with a cytokine.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, chemokines, and traditional polypeptide hormones.
  • cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin- associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor;
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroid hormone such as thyroxine
  • insulin proinsulin
  • relaxin pro
  • TGFs transforming growth factors
  • TGF-alpha and TGF-beta insulin-like growth factor-I and -II
  • EPO erythropoietin
  • osteoinductive factors interferons such as interferon- alpha, beta, and -gamma
  • colony stimulating factors CSFs
  • M- CSF macrophage-CSF
  • GM-CSF granulocyte-macrophage-CSF
  • G-CSF granulocyte-CSF
  • interleukins ILs
  • ILs interleukins
  • ILs interleukins
  • ILs interleukins
  • ILs such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12
  • IL-15 a tumor necrosis factor
  • TNF-alpha or TNF-beta and other polypeptid
  • genome edited CAR T cells redirected to a target cell e.g., a tumor or cancer cell
  • a target cell e.g., a tumor or cancer cell
  • the target cell expresses an antigen, e.g., a target antigen that is not substantially found on the surface of other normal (desired) cells.
  • the target cell is a bone cell, osteocyte, osteoblast, adipose cell, chondrocyte, chondroblast, muscle cell, skeletal muscle cell, myoblast, myocyte, smooth muscle cell, bladder cell, bone marrow cell, central nervous system (CNS) cell, peripheral nervous system (PNS) cell, glial cell, astrocyte cell, neuron, pigment cell, epithelial cell, skin cell, endothelial cell, vascular endothelial cell, breast cell, colon cell, esophagus cell, gastrointestinal cell, stomach cell, colon cell, head cell, neck cell, gum cell, tongue cell, kidney cell, liver cell, lung cell, nasopharynx cell, ovary cell, follicular cell, cervical cell, vaginal cell, uterine cell, pancreatic cell, pancreatic parenchymal cell, pancreatic duct cell, pancreatic islet cell, prostate cell, penile cell, gonadal cell, testis cell, hematopo
  • the target cell is solid cancer cell.
  • Illustrative examples of cells that can be targeted by the compositions and methods contemplated in particular embodiments include, but are not limited to those of the following solid cancers: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma,
  • GIST gastrointestinal stromal tumor
  • germ cell tumors glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,
  • nasopharyngeal cancer neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine
  • the target cell is liquid cancer or hematological cancer cell.
  • hematological cancers include, but are not limited to:
  • Illustrative examples of cells that can be targeted by the compositions and methods contemplated in particular embodiments include, but are not limited to those of the following leukemias: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocyte, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • myeloblastic promyelocyte
  • myelomonocytic monocytic
  • erythroleukemia hairy cell leukemia
  • HCL hairy cell leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myeloid le
  • cells that can be targeted by the compositions and methods contemplated in particular embodiments include, but are not limited to those of the following lymphomas: Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma and Non-Hodgkin lymphoma, including but not limited to B-cell non-Hodgkin lymphomas:
  • Burkitt lymphoma small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, marginal zone lymphoma, and mantle cell lymphoma; and T-cell non-Hodgkin lymphomas: mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, and precursor T-lymphoblastic lymphoma. .
  • Illustrative examples of cells that can be targeted by the compositions and methods contemplated in particular embodiments include, but are not limited to those of the following multiple myelomas: overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary
  • the target cell is a cancer cell, such as a cell in a patient with cancer.
  • the target cell is a cell, e.g., a cancer cell infected by a virus, including but not limited to CMV, HPV, and EB V.
  • a virus including but not limited to CMV, HPV, and EB V.
  • the target antigen is an epitope of alpha folate receptor, 5T4, ⁇ ⁇ ⁇ 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD 123, CD 138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRoc, GD2, GD3, Glypican-3 (GPC3), HLA- A1+MAGE1, HL A- A2+M AGE 1 , HL A- A3 +M AGE 1 , HLA-Al+NY-ESO-1, HLA- A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-l lRa
  • the genome edited CAR T cells manufactured by the compositions and methods contemplated herein provide improved adoptive cell therapy for use in the treatment of various conditions including, without limitation, cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency.
  • the specificity of a primary T cell is redirected to tumor or cancer cells by genetically modifying the primary T cell with a CAR contemplated herein.
  • the genome edited CAR T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient.
  • genome edited CAR T cells contemplated herein are used in the treatment of solid tumors or cancers.
  • genome edited CAR T cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical
  • teratoid/rhabdoid tumor basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer,
  • craniopharyngioma ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors,
  • DCIS ductal carcinoma in situ
  • GIST gastrointestinal stromal tumor
  • germ cell tumors glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,
  • nasopharyngeal cancer neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine
  • genome edited CAR T cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
  • genome edited CAR T cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung.
  • genome edited CAR T cells contemplated herein are used in the treatment of liquid cancers or hematological cancers.
  • genome edited CAR T cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
  • genome edited CAR T cells contemplated herein are used in the treatment of liquid cancers including, but not limited to the following leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblasts, lympho
  • methods comprising administering a therapeutically effective amount of genome edited T cells contemplated herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, are provided.
  • the cells are used in the treatment of patients at risk for developing a cancer.
  • particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer comprising administering to a subject in need thereof, a therapeutically effective amount of the genome edited T cells contemplated herein.
  • a method of treating a cancer in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising genome edited CAR T cells contemplated herein.
  • an effective amount e.g., therapeutically effective amount of a composition comprising genome edited CAR T cells contemplated herein.
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • the amount of T cells in the composition administered to a subject is at least 0.1 x 10 5 cells, at least 0.5 x 10 5 cells, at least 1 x 10 5 cells, at least 5 x 10 5 cells, at least 1 x 10 6 cells, at least 0.5 x 10 7 cells, at least 1 x 10 7 cells, at least 0.5 x 10 8 cells, at least 1 x 10 8 cells, at least 0.5 x 10 9 cells, at least 1 x 10 9 cells, at least 2 x 10 9 cells, at least 3 x 10 9 cells, at least 4 x 10 9 cells, at least 5 x 10 9 cells, or at least 1 x 10 10 cells.
  • the amount of T cells in the composition administered to a subject is at least 0.1 x 10 4 cells/kg of bodyweight, at least 0.5 x 10 4 cells/kg of bodyweight, at least 1 x 10 4 cells/kg of bodyweight, at least 5 x 10 4 cells/kg of bodyweight, at least 1 x 10 5 cells/kg of bodyweight, at least 0.5 x 10 6 cells/kg of bodyweight, at least 1 x 10 6 cells/kg of bodyweight, at least 0.5 x 10 7 cells/kg of bodyweight, at least 1 x 10 7 cells/kg of bodyweight, at least 0.5 x 10 8 cells/kg of bodyweight, at least 1 x 10 8 cells/kg of bodyweight, at least 2 x 10 8 cells/kg of bodyweight, at least 3 x 10 8 cells/kg of bodyweight, at least 4 x 10 8 cells/kg of bodyweight, at least 5 x 10 8 cells/kg of bodyweight, or at least 1 x 10 9 cells/kg of bodyweight.
  • compositions contemplated in particular embodiments may be required to effect the desired therapy.
  • a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
  • T cells can be activated from blood draws of from lOcc to 400cc.
  • T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, lOOcc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more.
  • using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.
  • compositions contemplated in particular embodiments may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • compositions are administered parenterally.
  • parenteral administration and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
  • compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.
  • a subject in need thereof is administered an effective amount of a composition to increase a cellular immune response to a cancer in the subject.
  • the immune response may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses.
  • Humoral immune responses mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced.
  • a variety of techniques may be used for analyzing the type of immune responses induced by the compositions, which are well described in the art; e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.
  • a method of treating a subject diagnosed with a cancer comprises removing immune effector cells from the subject, editing the genome of said immune effector cells and producing a population of genome edited immune effector cells, introducing a nucleic acid encoding a CAR into the cells, and administering the population of genome edited immune effector cells to the same subject.
  • the immune effector cells comprise T cells.
  • the methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo genome edited immune effector cells or on reintroduction of the genome edited progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells.
  • One method comprises genome editing peripheral blood T cells and introducing a nucleic acid encoding a CAR into the cells ex vivo and returning the transduced cells into the subject.
  • PBMCs peripheral blood mononuclear cells
  • VCN vector copy number
  • VCNs were similar for each donor for anti-BCMA CAR T cells cultured using the standard culture process (no EP), cells that were electroporated but received no mRNA (EP only), and cells electroporated with mRNA encoding a megaTAL that targets the TCRa gene (TCRa KO).
  • Figure 1 Cell Growth
  • Viable cell counts were performed on both donors during the 10 day culture process using a Nexcelom counting device and trypan blue exclusion to determine the impact of the electroporation protocol on the cell expansion.
  • Cells that were electroporated alone (EP only) or electroporated with TCRa megaTAL mRNA (TCRa KO) and incubated at 30°C overnight exhibited a delayed expansion relative to cells cultured using the standard process, but resumed similar growth kinetics between days 5 and 10 post PBMC culture initiation.
  • Figure 2
  • TCRa KO TCRa allele
  • ⁇ interferon-gamma
  • the biological activity of anti-BCMA CAR T cells with a disrupted TCRa allele (TCRa KO +/- Trex2) to BCMA-positive and BCMA-negative cell lines was assessed using a cytokine release assay. 5 x 10 4 T cells were expanded using the standard process (no EP), TCRa KO, or TCRa KO generated with Trex 2. Anti-BCMA CAR T cells were co-cultured T cells, or with BCMA positive and BCMA negative cell lines at a 1 : 1 ratio for 24 hours at 37°C.
  • Anti-BCMA CAR T cells manufactured using the standard process (no EP), EP only, and TCRa KO treatments described above were tested for in vivo efficacy using a Daudi tumor xenograft model.
  • 2 x 10 6 Daudi cells, which constitutively express luciferase were injected into NSG mice and allowed to engraft and grow until luciferase signals reached ⁇ 1 x 10 7 p/sec/cm 2 /sr (about 1 ldays post tumor cell injection) as assessed by a Xenogen IVIS imager.
  • TCRa KO cells across all doses, outperformed anti-BCMA CAR T cells cultured using the standard process and those which were
  • TCRa KO cells continue to outperform other anti-BCMA CAR T cells at day 43.
  • Figure 6B
  • Antigen dependent cytokine production in T cells transduced with a lentivirus encoding an anti-CD 19 CAR was compared to antigen dependent cytokine production in genome edited T cells containing an anti-CD 19 CAR integrated into the TCRa locus.
  • Human PBMCs (1 x 10 6 cells/mL) were activated with soluble anti-CD3 and anti- CD28 antibodies (50 ng/mL) on day 0. After 24hr incubation, 1 x 10 6 cells were transduced with an anti-CD19 CAR lentivims (LV-T cells; pBB146, SEQ ID NO: 8).
  • the lentiviral vector contained a CAR expression cassette comprising an MND promoter operably linked to a CAR comprising a CD8a-derived signal peptide, an anti-CD19 scFv, a CD8a derived hinge region and transmembrane domain, an intracellular 4-1BB co-stimulatory domain, and a ⁇ 3 ⁇ signaling domain.
  • Lentivims was prepared using established protocols. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et al. Nat. Protoc.
  • Adeno-associated vims (AAV) plasmids containing an anti-CD19 CAR was designed, constmcted, and verified (pBW1021, SEQ ID NO: 9).
  • the donor repair template contained a CAR expression cassette comprising an MND promoter operably linked to a CAR comprising a CD8a-derived signal peptide, an anti-CD 19 scFv, a CD8a derived hinge region and transmembrane domain, an intracellular 4- IBB co-stimulatory domain, and a ⁇ 3 ⁇ signaling domain.
  • the 5 ' and 3 ' homology regions of the donor repair template were about 650 bp each.
  • Recombinant AAV was prepared by transiently co-transfecting HEK 293 T cells with one or more plasmids providing the replication, capsid, and adenoviral helper elements necessary. rAAV was purified from the co-transfected HEK 293T cell culture using ultracentrifugation in an iodixanol-based gradient.
  • Human PBMCs (1 x 10 6 cells/mL) were activated with soluble anti-CD3 and anti- CD28 antibodies (50 ng/mL) on day 0.
  • cells were electroporated with buffer only (UTD control) or mRNA encoding a TCRa-targeting megaTAL polypeptide (SEQ ID NO: 11). Electroporated cells were incubated for one hour at 30°C and transduced with rAAV encoding an anti-CD 19 CAR donor repair template to drive homologous recombination (HR-T cells) at the TCRa locus.
  • the genome edited cells were incubated at 30°C for 24 hours and then were recovered and cultured at 37°C for 5 days with IL-2 containing medium.
  • Anti-CD 19 CAR Expression and TCRa Targeting Efficiency CDS negative cells
  • Cytotoxic potential of LV-T and HR-T cells was analyzed by co-incubating T cells with 50:50 mixtures of the CD19 + Nalm-6 (GFP) and K562 (BFP) target cells for 24 hours at an effector to target (E:T) ratio of 1 : 1.
  • the ratio of Nalm-6 vs. K562 following T cell co- culture is a direct readout of T cell cytotoxicity. Similar levels of CD 19 specific cytotoxicity was observed in both LV-T and FIR-T cell co-cultures.
  • Figure 8A The ratio of Nalm-6 vs. K562 following T cell co- culture is a direct readout of T cell cytotoxicity. Similar levels of CD 19 specific cytotoxicity was observed in both LV-T and FIR-T cell co-cultures.
  • Antigen dependent cytokine release was analyzed by co-culturing LV-T cells or FIR-T cells with CD19 + Nalm-6 cells for 24 hours at 1 : 1 E:T ratio. Culture supernatants were collected and IFNy release was analyzed using the Cytometric Bead Array (CB A) (BD biosciences). HR-T cells released increased amounts of IFNy compared to LV-T cells.
  • CB A Cytometric Bead Array
  • Antigen dependent cytokine production in T cells transduced with a lentivirus encoding an anti-CD 19 CAR was compared to antigen dependent cytokine production in genome edited T cells containing an anti-CD 19 CAR integrated into the TCRa locus.
  • Human PBMCs (1 x 10 6 cells/mL) were activated with soluble anti-CD3 and anti-
  • CD28 antibodies (50 ng/mL) on day 0.
  • 1 x 10 6 cells were transduced with an anti-CD19 CAR lentivirus (LV-T cells; pBB146, SEQ ID NO: 8).
  • the lentiviral vector contained a CAR expression cassette comprising an MND promoter operably linked to a CAR comprising a CD8a-derived signal peptide, an anti-CD 19 scFv, a CD8a derived hinge region and transmembrane domain, an intracellular 4-lBB co-stimulatory domain, and a 0)3 ⁇ signaling domain.
  • Lentivirus was prepared using established protocols. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et al. Nat. Protoc.
  • Adeno-associated virus (AAV) plasmids containing an anti-CD19 CAR was designed, constructed, and verified (pBW400, SEQ ID NO: 13).
  • the donor repair template contained a CAR expression cassette comprising an MND promoter operably linked to a CAR comprising a CD8a-derived signal peptide, an anti-CD 19 scFv, a CD8a derived hinge region and transmembrane domain, an intracellular 4- IBB co-stimulatory domain, and a 0)3 ⁇ signaling domain, and a woodchuck post-transcriptional regulatory region (WPRE).
  • the 5 ' and 3 ' homology regions of the donor repair template were about 650 bp each.
  • Recombinant AAV was prepared by transiently co-transfecting HEK 293 T cells with one or more plasmids providing the replication, capsid, and adenoviral helper elements necessary. rAAV was purified from the co-transfected HEK 293T cell culture using ultracentrifugation in an iodixanol-based gradient.
  • Human PBMCs (1 x 10 6 cells/mL) were activated with soluble anti-CD3 and anti- CD28 antibodies (50 ng/mL) on day 0.
  • cells were electroporated with buffer only (UTD control) or mRNA encoding a TCRa-targeting megaTAL polypeptide (SEQ ID NO: 11). Electroporated cells were incubated for one hour at 30°C and transduced with rAAV encoding an anti-CD 19 CAR donor repair template to drive homologous recombination (HR-T cells) at the TCRa locus.
  • the genome edited cells were incubated at 30°C for 24 hours and then were recovered and cultured at 37°C for 5 days with IL-2 containing medium.
  • Cytotoxic potential of LV-T and HR-T cells was analyzed by co-incubating T cells with 50:50 mixtures of the CD19 + Nalm-6 (GFP) and K562 (BFP) target cells for 24 hours at an effector to target (E:T) ratio of 1 : 1.
  • the ratio of Nalm-6 vs. K562 following T cell co- culture is a direct readout of T cell cytotoxicity. Similar levels of CD 19 specific cytotoxicity was observed in both LV-T and FIR-T cell co-cultures.
  • Figure 10A The ratio of Nalm-6 vs. K562 following T cell co- culture is a direct readout of T cell cytotoxicity. Similar levels of CD 19 specific cytotoxicity was observed in both LV-T and FIR-T cell co-cultures.
  • Antigen dependent cytokine release was analyzed by co-culturing LV-T cells or FIR-T cells with CD19 + Nalm-6 cells for 24 hours at 1 : 1 E:T ratio. Culture supernatants were collected and IFNy, IL-2, IL-4, and TNFa release were analyzed using the Cytometric Bead Array (CBA) (BD biosciences). HR-T cells released increased amounts of cytokines compared to LV-T cells. Figure 10B. EXAMPLE 4
  • T cell exhaustion in T cells transduced with a lentivirus encoding an anti-CD 19 CAR was compared to T cell exhaustion in genome edited T cells containing an anti-CD 19 CAR integrated into the TCRa locus.
  • AAV adeno-associated virus
  • Recombinant AAV was prepared by transiently co-transfecting HEK 293 T cells with one or more plasmids providing the replication, capsid, and adenoviral helper elements necessary.
  • rAAV was purified from the co-transfected HEK 293T cell culture using ultracentrifugation in an iodixanol-based gradient.
  • the lentiviral vector contained a CAR expression cassette comprising an MND promoter operably linked to a CAR comprising a CD8a-derived signal peptide, an anti-CD 19 scFv, a CD8a derived hinge region and transmembrane domain, an intracellular 4- IBB co- stimulatory domain, and a CD3 ⁇ signaling domain.
  • Lentivirus was prepared using established protocols. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
  • Primary human T cells were activated with CD3 and CD28, as described in Example 1. Activated primary human T cells were electroporated with in vitro transcribed megaTAL mRNA and transduced with rAAV targeting vectors encoding the anti-CD 19 CAR transgene (FIR-CAR T cells); or activated primary human T cells were transduced with a lentivirus encoding an anti-CD 19 CAR (LV-CAR T cells).
  • LV-T and HR-T cells were co-cultured with CD 19 expressing Nalm-6 cells in 1 : 1 Effector (E) cell to Target (T) cell ratio.
  • T cell exhaustion marker expression (PD-Ll, PD-1, and Tim-3) was measured at 24 hours and 72 hours of co-culture.
  • FIR-CAR T cells showed reduced upregulation of PD-1 and PD-Ll compared to LV-CAR T cells.
  • Figure 11 A At 72 hours, FIR-CAR T cells showed reduced upregulation of PD-1 and Tim-3 compared to LV-CAR T cells.
  • Figure 1 IB Figure 1 IB.

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Abstract

L'invention concerne des compositions améliorées pour des thérapies par cellules effectrices immunes adoptives pour le traitement, la prévention ou le soulagement de nombreux états pathologiques dont, entres autres, le cancer, les maladies infectieuses, les maladies auto-immunes, les maladies inflammatoires et l'immunodéficience.
PCT/US2017/026602 2016-04-07 2017-04-07 Compositions de lymphocytes t récepteurs d'antigènes chimériques WO2017177137A1 (fr)

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CA3020330A CA3020330A1 (fr) 2016-04-07 2017-04-07 Compositions de lymphocytes t recepteurs d'antigenes chimeriques
AU2017248259A AU2017248259A1 (en) 2016-04-07 2017-04-07 Chimeric antigen receptor T cell compositions
US16/092,146 US20190161530A1 (en) 2016-04-07 2017-04-07 Chimeric antigen receptor t cell compositions
CN201780031902.3A CN109415687A (zh) 2016-04-07 2017-04-07 嵌合抗原受体t细胞组合物
JP2018552793A JP2019510503A (ja) 2016-04-07 2017-04-07 キメラ抗原受容体ｔ細胞組成物
EP17779910.3A EP3440191A4 (fr) 2016-04-07 2017-04-07 Compositions de lymphocytes t récepteurs d'antigènes chimériques

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US201662319703P 2016-04-07 2016-04-07
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US20190161530A1 (en) 2019-05-30
EP3440191A4 (fr) 2019-12-11

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